An activation antigen on a subpopulation of B lymphocytes identified by the monoclonal antibody CMRF-17.

The identification of membrane molecules expressed on subpopulations of B lymphocytes is of potential significance because these molecules may be candidates for regulating the activation, proliferation and differentiation of B cells. A new monoclonal antibody, CMRF-17, which reacts with a subpopulation of tonsil B lymphocytes has been produced. The antibody did not react with T lymphocytes in tonsil or peripheral blood nor most peripheral blood B lymphocytes but did label erythrocytes and some platelets. In tonsil, the germinal centre cells, cells in the interfollicular region and endothelial cells were positive, but mantle zone B cells were negative. Double labelling experiments showed that CMRF-17 reacted with activated tonsillar lymphocytes. The antigen recognized by CMRF-17 was heat stable, resistant to treatment with proteolytic enzymes and neuraminidase and was shown to be a carbohydrate determinant on one or more glycolipids. These characteristics of the antigen recognized by CMRF-17 and its pattern of reactivity distinguish this antibody from other monoclonal antibodies recognizing B-cell activation markers. It was notable that of the B-lymphoid malignancies tested to date, including those of probable follicular origin, few stained with CMRF-17.     

A novel human B-lymphocyte antigen shared with lymphoid dendritic cells: characterization by monoclonal antibody.

A novel cell-surface antigen (L25) expressed on human B cells was identified using a B cell-reactive monoclonal antibody (TB1-4D5). This L25 antigen was expressed on most B-lineage cells but not other cell types including thymocytes, T cells, granulocytes and monocytes. Thus, L25 existed on the majority of normal B cells present in the blood and lymphoid tissues, on cultured cell lines derived from normal and malignant B cells, and on neoplastic cells isolated from patients with B cell-derived malignancies. Though L25 was persistently expressed on B cells until 7 days after their activation with pokeweed mitogen (PWM), neither normal nor neoplastic plasma cells expressed L25. Moreover, L25 was present on cultured as well as freshly isolated leukaemic cells with common acute lymphatic leukaemia (CALL) antigen, which have been thought to correspond to the early B-cell ontogeny. Besides pan-B cell reactivity of TB1-4D5 antibody, it apparently cross-reacted with so-called dendritic or interdigitating cells located in the thymic-dependent areas of peripheral lymphoid organs, which have been presumably ascribed to those associated with accessory-cell function. Functional studies showed that anti-L25 (TB1-4D5) antibody had inhibitory effect on induction of immunoglobulin synthesis by PWM-stimulated B cells.     

Generation and characterization of a monoclonal antibody that recognizes an activation antigen expressed by a majority of adherent lymphokine-activated killer cells

In an attempt to generate murine natural killer (NK) cell-specific monoclonal antibodies (MAbs) by immunizing Balb/c mice with C57BL/6 (B6) A-LAKs, we have isolated a hybridoma, CS/NicT.2, which secretes an IgM that recognizes a majority of B6 and B6 Rag-1-/- A-LAKs. The CS/NicT.2 antigen is highly expressed by A-LAKs, but only at extremely low levels on resting splenocytes, suggesting that its expression is tightly associated with IL-2 activation. Among the cell lines examined, only CTLL-2 expresses the CS/NicT.2 antigen at relatively high levels. A low level of CS/NicT.2 staining is also detected on resting allo-specific cytotoxic T lymphocytes (CTL) clones, AB.1 and C11. In addition, a similar low level of CS/NicT.2 staining is detected on the T-helper cell line HT-2. The CS/NicT.2 antigen is upregulated by ionomycin but not by phorbol 12-myristate 13-acetate (PMA). For the CTL clones examined, CS/NicT.2 staining is also dramatically increased by anti-TCRbeta or anti-CD3epsilon stimulation. Protease treatments of CTLL-2 show that this antigen is proteinase K sensitive, but relatively resistant to trypsin digestion. Furthermore, the CS/NicT.2 antigen exhibits a relatively fast turnover rate as assessed by proteinase K and cycloheximide treatments of CTLL-2, suggesting that the CS/NicT.2 antigen may have a short half-life on the cell surface.     

Characterization of an antigen defined by monoclonal antibody KMO1.

We have isolated an antigen defined by the monoclonal antibody KMO1 from tissue culture supernatant as a glycoprotein and from cancer cells as a glycolipid. The antigenic determinant was a carbohydrate. Antibody KMO1 inhibited the binding activity of 19-9 to CA19-9, but 19-9 antibody did not inhibited the binding activity of KMO1 to KMO1 antigen. The glycolipid antigen of KMO1 was a monosialoganglioside and was separated into 3 components on thin layer chromatography. The 19-9 antibody reacted with one of the components. These results suggest that the KMO1 antigen is similar to but not identical to CA19-9.     

OX48, a monoclonal antibody against a 70,000 MW rat activation antigen expressed by T cells bearing the high-affinity interleukin-2 receptor.

The monoclonal antibody (mAb) OX48 recognizes a 70,000 MW cell-surface protein present in a small percentage of activated rat T cells and in CD8+ rat x BW5147 interleukin-2 (IL-2)-dependent T-cell hybridomas, but not in resting spleen cells or in IL-2-independent T-cell hybrids. OX48 antibody added simultaneously with concanavalin A (Con A) to resting spleen cells inhibits the cell proliferation and reduces the IL-2 production. However, addition of IL-2 does not restore the mitogenic response. Growth of rat blast T cells or IL-2-dependent hybrids is not affected by the OX48 antibody. There is a close correlation between the expression of high-affinity IL-2 receptors (IL-2R) and the OX48 antigen in T-cell hybridomas. In spite of this striking correlation, OX48 mAb does not inhibit the binding of 125I-IL-2 to the IL-2-dependent hybrids, and is unable to immunoprecipitate any of the proteins chemically cross-linked to 125I-IL-2. Therefore, the OX48 molecule represents a new rat activation antigen, undefined in other species, and probably involved in the early steps of T-cell activation.     

Characterization of the specificity by immunohistology of a monoclonal antibody to a novel epithelial antigen of ovarian carcinomas

An immunohistological study, using the avidin-biotin-peroxidase complex method, was carried out to define the reactivity profile of a murine monoclonal antibody, MOv2, which recognizes a novel glycoprotidic antigen associated with ovarian epithelial tumors. Among the primary ovarian tumors tested, MOv2 immunostained 93% of mucinous and 75% of serous cystadenomas, 100% of mucinous, 81% of serous and 73% of endometrioid carcinomas. Undifferentiated and clear cell tumors revealed more limited reactivity with the antibody, whereas ovarian sex cord-stromal and germinal tumors were immunonegative. Positive reactions were also documented in omental metastases from primary ovarian carcinomas. No immunoreactivity was detected in normal ovarian epithelium, whereas the cells lining Walthard's nests adjacent to the fallopian tubes and a variety of normal epithelia were consistently immunolabeled. These included the lining epithelia of the gastrointestinal tract, bronchi and endocervix, and the epithelium of salivary, biliary and pancreatic ducts and sweat glands. To a lesser extent, positive reactions were detected in other surface epithelia, such as squamous and transitional epithelia. Among tumors other than ovarian, MOv2 consistently reacted with adenocarcinomas and squamous cell carcinomas from different sites, most notably breast, lung and gastrointestinal tract, and with transitional cell carcinomas. In contrast, no staining was demonstrated in non-epithelial malignancies. The antigen defined by MOv2 may be operationally useful as a marker of epithelial lineage in tumor histopathology. Its pattern of immunohistochemical distribution indicates that an antigenic phenotype shared by normal surface epithelia and non-ovarian carcinomas is strongly associated with common epithelial neoplasms of the ovaries.     

The monoclonal antibody 11G7 recognizes a novel differentiation antigen expressed on hemopoietic precursor cells

A monoclonal antibody (11G7) detecting a novel antigen on human hemopoietic progenitor cells (named 11G7R = 11G7 receptor) was raised by immunization of a Balb/c mouse with the leukemic blasts of a patient suffering from chronic myelogenous leukemia blast crisis (CML-BC). The antigen is expressed on most of MHC class II bearing peripheral blood leucocytes (PBL) and on a subpopulation of bone marrow mononuclear cells (BMMNC). By FACS-sorting and colony assays, it could be demonstrated that 11G7R is expressed on myelo-monocytic and myelo-granulocytic bone marrow precursor cells (GM-CFC, G-CFC, M-CFC) but is absent from erythroid precursor cells (BFU-E) and on cells exhibiting the capacity to form mixed colonies (GEMM-CFC). Double-fluorescence analysis on BMMNC revealed that 11G7R is expressed on a subset of B-cells, myeloid cells and cells carrying the HPCA-1 antigen (CD34). It has a similar distribution pattern to the myeloid antigens CD13 and CD33. However, in contrast to these antigens, 11G7R is also expressed on the blasts of several lymphoid leukemias (4/9 B-ALL, 1/2 T-ALL) and therefore it is not restricted to the myeloid lineage.     

CNA.42, a new monoclonal antibody directed against a fixative-resistant antigen of follicular dendritic reticulum cells.

A new monoclonal antibody (MAb), CNA.42, was generated using the CEM T-cell line. It recognizes a 120-kd formalin-resistant glycosylated antigen that is mainly expressed by follicular dendritic reticulum cells (FDRCs). This antigen is also expressed by a few mononuclear cells in the paracortical area of reactive lymph nodes and by some cortical thymocytes. Two hundred and eighty-nine cases of hematopoietic tumors of various types were tested with this antibody. They showed either intact FDRC networks or FDRC networks dispersed among malignant cells. In follicular lymphomas, the follicular pattern was highlighted by CNA.42 MAb. Expanded FDRC networks were found in angioimmunoblastic T-cell lymphomas. Neoplastic cells were positive in 43.6% (24/55) of T-cell and 4.6% (6/129) of B-cell lymphomas. The highest percentage of cases with positive neoplastic cells was found in anaplastic large-cell lymphomas (62.5%; 15/24). In Hodgkin's disease, FDRC networks, sometimes encasing Hodgkin and Reed-Sternberg (HRS) cells, were found. HRS cells were also stained by this antibody in 23 (21.9%) of the 105 cases examined. A variety of normal nonlymphoid tissues and nonhematopoietic tumors, such as some neurogenic tumors, carcinoma, and occasional sarcomas, were found to be positive. Analysis of the reactivity of CNA.42 antibody with FDRCs of lymphoid tissue from different animal species showed similar reactivity to that observed in humans, suggesting widespread evolutionary conservation of the antigen recognized by this antibody. In daily diagnostic practice, CNA.42 MAb seems to be a suitable FDRC marker and possibly has an auxiliary role in recognizing T-cell lymphomas.     

Modulation of human B cell responses by a monoclonal antibody to an activation antigen 4F2.

Antigen specific human antibody responses can be modulated in vitro by the addition of 4F2 antibody, a monoclonal antibody (MoAb) which recognizes an antigen on activated T cells and B cells. Specific antibody responses induced with the antigen are suppressed by the addition of 4F2. However, specific antibody responses induced with the polyclonal activator, pokeweed mitogen (PWM), are significantly enhanced by the addition of 4F2. Proliferative responses to both antigen and PWM are suppressed by the addition of 4F2. The enhancement of PWM stimulated responses by 4F2 is mediated by T cells. However, in the absence of T cells, 4F2 can directly inhibit antigen specific B cells. Polyclonal Ig production stimulated by PWM was also enhanced by 4F2. Thus, the immunomodulating effects of the 4F2 MoAb are the result of a balance of enhancement and suppression mediated at the T cell and the B cell level, respectively.     

Characterization of immature B cells by a novel monoclonal antibody,by turnover and by mitogen reactivity

The transit of immature to mature sIgM⁺ B cells, the life span, maturation kinetics and response to polyclonal activators have been analyzed with the help of a new mAb (493), that distinguishes immature, 493⁺ from mature, 493⁻ B cells in a variety of mouse strains tested. Analysis of the turnover of immature 493⁺ B cells by bromodeoxyuridine (BrdU) labeling kinetics indicate that only 10 – 20 % of the cells reach the spleen as immature 493⁺ cells. The life span of 493⁺ B cells in bone marrow and spleen is around 4 days. BrdU chase experiments show that most of the immature cells in spleen enter the pool of mature, 493⁻ B cells where they gain a longer life span of 15 – 20 weeks. Immature and mature B cells respond equally well to LPS stimulation; anti-CD40, however, stimulates mature B cells better than immature B cells. IgM cross-linking of mature B cells results in proliferation, while it induces apoptosis in immature B cells. This apoptosis of immature cells can be inhibited by co-stimulation with anti-CD40 or by overexpression of bcl-2. We speculate that Ig receptor ligand-mediated apoptosis (negative selection) plays a major role in the transit of immature B cells from bone marrow to spleen, but only a minor role in the transit from immature B cells to mature B cells in the spleen.     

A monoclonal antibody to a determinant of the rat T cell antigen receptor expressed by a minor subset of T cells

A hybridoma producing the monoclonal antibody HIS42 was isolatedfrom a fusion between spleen cells from a BALB/c mouse immunizedwith rat thymocytes and the fusion partner SP2/0. This antibodyrecognizes a minor subset of T cells in every haplotype of rattested so tar. The subpopulation of HIS42 positive T cells containsboth CD4+ and CD8+ cells, in the same ratio as found in theperipheral T cell population. When bound to Sepharose beads,HIS42 induces T cell proliteration in the presence of Interleukin-2.In contrast to lymph node T cells, a number of thymocytes werefound to express HIS42 only in the cytoplasm or together withmembrane expression. Most bright HIS42 surface labelled thymocyteswere also positive for MRC OX-44, a marker predominantly identifyingmature thymocytes. SDS-PAGE analyses of the membrane moleculeslmmunoprecipitated by HIS42 show two bands on unreduced gels.One of these bands (85 kd) runs as two separate bands at 35and 48 kd on reduction. The other much weaker broad band ({smalltilde}100 kd) is hardly affected by reduced conditions. Takentogether these data suggest that HIS42 is directed against adeterminant on the rat T cell receptor for antigen, which iscommon to a small number of T cells.     

D11, a novel monoclonal antibody specific for human mature macrophages and peripheral blood monocytes.

A new monoclonal antibody designated Mab D11 is described, which shows a restricted reactivity to cells of the monocyte/macrophage system. When tested by light and electron microscopic immunoperoxidase methods, Mab D11 specifically reacts with blood monocytes and stains resident macrophages in a wide variety of human tissues; it does not mark the macrophages of other species, i.e., rat, swine and mouse. Antigen-presenting cells, e.g., Langerhans cells, are Mab D11 negative. Mab D11 reveals the antigen on cryostat and paraffin tissue sections. Ultrastructurally the antigen recognized by Mab D11 in all macrophage types studied is located on the plasma membrane and within cytoplasmic structures including lysosomes. On immunoblotting, Mab D11 detects the 125-kDa antigen in human liver and the 135-kDa protein in tumours of histiocytic origin. The similarity of Mab D11 to known "pan-macrophage" monoclonal antibodies is discussed.     

Characterization of a glial associated antigen GA-1 by monoclonal antibody

A glial antigen (GA-1) was identified by monoclonal antibodies (MAb) raised against C6 rat glioma cells. MAb-7D3 (IgG_2aκ) revealed GA-1 as a single protein band with a R_f value of 0.09 by the use of basic-PAGE Western blot. SDS-PAGE Western blot and radioimmunoprecipitation (RIP) further resolved Ga-1 into two subunits with a molecular weight of 200 and 78 kDa respectively. Subcellular localization by immunocytochemical staining revealed its cytosolic presence with a punctate pattern perinuclearly. Significant expression of GA-1 may be detected in 4 glioma or glial cell lines derived from rat brain. However, no expression may be detected in the rest of the 18 mammalian cell lines or primary neural cell cultures examined. All of the above data thereby suggest that GA-1 may be glial specific whereas the epitope of GA-1 defined by MAb-7D3 is species (rat) specific.     

Characterization of a strain-specific monoclonal antibody to hepatitis delta virus antigen

Sequences of the hepatitis delta virus (HDV) vary to different degrees among isolates. A monoclonal antibody, designated as HP6A1, against the antigen of HDV (HDAg) has been characterized for its specificity. HP6A1 bound to HDAg of isolate 25 (genotype I) that was used for immunization, but not to others of both genotypes I and II. The epitope recognized by HP6A1 was then determined by a phage library displaying various heptapeptides. A consensus peptide deduced has the best match with that of residues 4-10 of HDAg (isolate 25). To confirm the phage mapping result, Escherichia coli recombinant proteins containing different lengths and various segments of HDAg (isolate 25) were constructed. The shortest HDAg segment contained in the fusion protein that reacted with HP6A1 was residues 1-10. When this peptide was added to the N-terminus of a heterologous protein engineered for eucaryotic expression, the fusion protein was detected by HP6A1. It is concluded that HP6A1 recognizes an epitope located at the N-terminus of HDAg (isolate 25). Since viruses of quasi-species exist in natural infections, a question of how different viral strains interact in vivo remains to be explored. The highly specific MAb opens a possibility to examine the fate of one strain in the presence of other related species in a cell transfection system.     

Epitopic heterogeneity of the CD36 antigen expressed by normal and neoplastic endothelial cells. An immunohistochemical study with a novel monoclonal antibody 8C9.

Phenotypic and functional heterogeneity of endothelial cells (ECs) is being recognized with increasing frequency. Here we report a novel murine monoclonal antibody (MoAb), named 8C9, that detects a unique epitope on the leukocyte differentiation antigen CD36 (platelet glycoprotein IV or IIIb) expressed by both normal and neoplastic ECs. In immunohistochemical and flow-cytometric studies, 8C9-immunoreactivity was detected on capillary ECs, adipocytes, monocytes, platelets and a human monocytoid cell line U-937, which are known to express the CD36 antigen. Blocking experiments using U-937 cells and studies on cryostat sections revealed that a murine MoAb OKM5, which detects the CD36 antigen, blocked the binding of 8C9 to its antigen. Immunoblot analysis showed that 8C9 bound to a 97-kDa membrane protein expressed by U-937 cells treated with phorbol ester. These results indicate that 8C9 detects the CD36 antigen. However, the findings that some OKM5-positive normal ECs in the liver, spleen and lymph nodes as well as neoplastic ECs in a cutaneous angiosarcoma did not react with 8C9, together with the fact that the CD36 antigen does not form a complex or associate with other proteins, suggest epitopic heterogeneity of the CD36 antigen expressed by these tissues.     

Follicular dendritic cells in bone marrow lymphoproliferative diseases: an immunohistochemical study including a new paraffin-resistant monoclonal antibody, DR53

Two-hundred and twenty-one bone marrow biopsies with lymphoid infiltrates were studied histologically and immunohistochemically, to assess the incidence and the pattern of follicular dendritic cells. Three monoclonal antibodies selective for follicular dendritic cells were used: CD21, CD35 and DR53, all reactive on paraffin-embedded material. Follicular dendritic cells were present in two of 38 benign lymphoid aggregates, 92 of 134 low grade B-cell lymphomas (45 of 62 lymphocytic, 16 of 27 lymphoplasmacytoid, 0 of six hairy cell leukaemias, five of six centrocytic, 19 of 21 centroblastic-centrocytic, seven of 12 low grade NOS), one of 23 high grade B-cell lymphomas, 0 of 10 T-cell lymphomas, 0 of three Hodgkin's disease and four of 13 suspicious infiltrates. Follicular dendritic cells were found in lymphomatous involvement with nodular, patchy and massive growth pattern, but not in interstitial ones. They formed follicle-like networks, whose number and size were directly correlated to the tumour mass. The origin and frequency distribution of follicular dendritic cells in bone marrow biopsy lymphomas is discussed and the diagnostic relevance of follicular dendritic cell immunostaining in routine bone marrow biopsy lymphoid infiltrates is assessed.     

Characterisation of normal peripheral blood cells in cycle identified by monoclonal antibody Ki-67.

AIMS: To investigate the numbers, morphology, and lineage assignment of Ki-67 positive cells in peripheral blood from normal subjects. METHODS: Single and double immunoenzymatic staining procedures, immunoperoxidase, and immunoalkaline phosphatase were used with Ki-67, a monoclonal antibody that recognises a nuclear antigen present in proliferating cells, with markers expressed in B and T lymphocytes and monocytes. RESULTS: In the five healthy donors 2.1% (range 1.6-3.7%) cells of the blood mononuclear fraction and 2.7% (range 2.3-3.9%) lymphocytes were Ki-67 positive. Of these, 88% (range 85-90%) were small cells and 12% (range 10-15%) were medium sized. Forty one per cent of the Ki-67 positive cells were CD3 positive by double immunoenzymatic staining and corresponded to T lymphocytes, and 11.4% were mature B cells expressing kappa or lambda light chains. Monocytes detected by the anti-lysozyme antibody were consistently Ki-67 negative. Half of the Ki-67 positive lymphocytes could not be accounted for by B or T cells with the markers used. Most Ki-67 positive cells were of small size; the B lymphocytes in cycle showed abundant cytoplasm and features suggestive of lymphoplasmacytic differentiation. CONCLUSIONS: The methodology described is useful for the simultaneous detection of nuclear and cytoplasmic antigens. The demonstration that a proportion of normal blood lymphocytes are in cell cycle raises the issue of whether immunophenotypic analysis of Ki-67 positive cells in haemopoietic malignancies with peripheral blood disease should be carried out to define more precisely the proportion of normal and neoplastic cells in cycle.