Marginal zone macrophages in the mouse spleen identified by a monoclonal antibody. Anatomical correlation with a B cell subpopulation

The reactivity of a monoclonal antibody, ER-TR9, that demonstrates heterogeneity among mononuclear phagocytes is described. In the spleen, ER-TR9 exclusively reacts with a population of macrophages located in the marginal zone. ER-TR9 does not react with macrophage antigen 1-positive red pulp macrophages or any other types of splenic stromal cells. ER-TR9+ ve cells localize in anatomical proximity of a subpopulation of B cells, i.e., B cells that are immunoglobulin M positive and weakly positive to negative for immunoglobulin D. The possible significance of this particular interaction between both cell types during the immune response is discussed.     

Expression of C3d receptors during human B cell differentiation: immunofluorescence analysis with the HB-5 monoclonal antibody

We have examined human B lymphocytes at different stages of differentiation for the expression of surface receptors for the C3d fragment of complement. C3d receptors (C3dR) were identified by indirect immunofluorescence using the HB-5 monoclonal antibody, which recognizes a 145,000 m.w. C3dR molecule on B lymphocytes. Pre-B and immature B cells from fetal bone marrow and liver did not express C3dR, whereas a small subpopulation (25%) of B cells in fetal spleen were C3dR+. Approximately 50% of the B cells in adult bone marrow were C3dR+, whereas the more mature B cells in the blood of newborns and adults and in peripheral lymphoid tissue of adults uniformly expressed the C3dR. Activated B cells responsive to T cell-derived differentiation factors were C3dR+, whereas plasma cells rarely expressed C3dR. T cells, NK cells, erythrocytes, and myelomonocytic cells did not express detectable surface C3dR. These results suggest that in hematopoietic and lymphoid tissues, the expression of C3dR is a specific feature of relatively mature lymphoid cells of B lineage.     

Basally located epithelial cell surface component identified by a novel monoclonal antibody technique

Tubular aggregates of glandular epithelial cells (gland fragments) were isolated from human endometrium by collagenase digestion of surrounding stroma, thus exposing the basal surfaces of the cells. Using these aggregates as immunogen, monoclonal antibodies could be derived that recognized basally located antigens. One such antibody, G71, is described, that binds to a basal epithelial cell antigen present in a variety of human epithelia. Epitope-bearing molecules in the range Mr 60 000-180 000 are present in two of the tissues studied, amnion and endometrium. The epitope is associated with areas of epithelial cell-extracellular matrix contact.     

A subpopulation of trypanosome microtubules recognized by a monoclonal antibody to tubulin

Two hybridoma cell lines were selected after the fusion of the myeloma cell line X-63 Ag8-653 with spleen cells from mice immunized with bovine brain microtubules. These lines, clones 3F3 and 16D3, secrete IgM antibodies both staining a fibrillar network in fibroblasts. Autoradiography of immunoblots of SDS gels showed that the antigenic determinants defined by these antibodies are present on tubulin and also on several other polypeptides in mammalian cells. In contrast, they were found to react only with tubulin in Trypanosoma brucei, parasitic protozoan which are the causative agent of sleeping sickness. By immunofluorescence microscopy, 3F3 bound only to a subpopulation of microtubules associated with the flagellum of these cells when, under the same conditions, 16D3 stained other microtubule populations including sub-pellicular microtubules. These results show that flagellar tubulin differs from tubulin of other locations in the same cell by at least one antigenic determinant which could be involved in microtubule specialization.     

Suppressor cell function of human granular lymphocytes identified by the HNK-1 (Leu 7) monoclonal antibody

The HNK-1 (Leu 7) differentiation antigen defines a subpopulation of human granular lymphocytes with natural killer (NK) and K cell function. In this study, we investigated whether HNK-1+ cells, identified with the monoclonal antibody and purified with a fluorescence-activated cell sorter (FACS), could function as suppressor cells. The results demonstrated that purified HNK-1+ cells efficiently suppressed both PWM-induced IgG production by B cells and T cell proliferation in mixed lymphocyte reactions (MLR). Manifestation of this suppressor cell activity required immune complex activation and was partially sensitive to 2000 rad irradiation. This suppressor cell activity was predominantly mediated by a subset of HNK-1+ cells that have previously been shown to have maximum NK function and lack expression of the E rosette (ER) receptor and T cell antigens (e.g., T3 and T8). Thus, HNK-1+ER- cells suppressed a MLR by an average 52%; HNK-1+ER+ were one-half as efficient, causing an average 23% suppression. For comparison, we also examined the characteristics of Leu 2a+ suppressor T lymphocytes. In contrast to HNK-1+ cells, unactivated Leu 2a+ cells suppressed both B and T cell responses. This suppressor activity was not augmented by immune complex activation and was absolutely radio-sensitive in PWM assays. HNK-1+ cells, especially the HNK+ER- subset, can therefore mediate suppressor cell function in addition to their spontaneous cytotoxic function. Furthermore, some of their suppressor cell properties are distinct from those attributed to other types of suppressor lymphocytes.     

Cytotoxic murine monoclonal antibody recognizing an ovine lymphocyte subpopulation similar to the human OKT4-positive set

A cytotoxic murine immunoglobulin G2b monoclonal antibody was produced from immunization with ovine thymocytes. It reacts with a monomorphic determinant on ovine lymphocytes. This antibody 11.2 G11 does not react with B cells, lyses 50 to 60% of peripheral blood T cells, and precipitates a single chain protein with an apparent m.w. of 57,000. Its effect on mitogen- and antigen-driven lymphocyte proliferation supports its similarity in the sheep to the OKT4 antibody in humans.     

Delineation and characterization of human B cell subpopulations at various stages of activation by using a B cell-specific monoclonal antibody

Human tonsillar B cells were separated into three distinct subpopulations, Ba-/IgD+, Ba+/IgD+, and Ba+/IgD-, by using a B cell-specific monoclonal antibody (anti-Ba) that recognizes only activated B cells, and anti-IgD antibody. Stimulation of Ba-/IgD+ cells with anti-mu plus PHA-conditioned culture supernatant (PHA-sup) or TPA induced Ba+/IgD+ cells, which reverted to Ba-/IgD+ phenotype in the absence of continuous stimulation. Further stimulation of Ba+/IgD+ cells with several B cell activators, such as TPA plus anti-mu or PWM plus T cells, resulted in the loss of IgD expression. Three-color FACS analysis showed that the expression of transferrin receptor (TFR) was at its maximum in Ba+/IgD- cells, and the intensity of this expression was proportional to that of Ba expression in Ba+/IgD+ cells. PHA-sup induced maximum proliferation in Ba+/IgD- cells, and the degree of response was a function of the intensity of Ba expression in Ba+/IgD+ cells. PHA-sup or purified BCDF (BSF-2) induced Ig secretion preferentially in Ba+/IgD- cells. Taken together, these results show that resting B cells (Ba-/IgD+) are activated into Ba+/IgD+ cells, and then into Ba+/IgD- cells, under mitogenic stimulation, and BCDF induces the final maturation of Ba+/IgD- cells into Ig-secreting cells. Ba+/IgD- cells, which maximally expressed TFR as well as Ba and displayed maximum proliferative response to PHA-sup, did not express any Tac antigen. On the other hand, in vitro activated B cells expressed Ba and TFR as well as Tac antigen.     

Molecular profile, tissue distribution and prognostic evaluation of a human melanoma-carcinoma antigen recognized by the murine monoclonal antibody B1.1

Using the murine monoclonal antibody (MoAb) B1.1 we have analyzed the immunochemical profile and the tissue distribution of a human melanoma associated antigen (MAA) carrying an epitope shared by the 180 kd CEA. Results of this study have demonstrated that the epitope expressed by the MAA is carried by a distinct set of molecules of 110-140 kd. Similarly to the 180 kd CEA molecules synthesized by carcinomas, the expression of the melanoma associated CEA like components (MA-CEA) is upregulated by IFN-alpha. The tissue distribution of MA-CEA is not restricted to malignant primary and metastatic melanocytic lesions but is found also at low levels in 64% of benign nevi. No circulating CEA was found in patients bearing widespread metastatic disease of MA-CEA positive lesions. Preliminary clinical evaluation of stage I melanoma patients bearing MoAb B1.1 positive lesions has not shown a significative prognostic association of this phenotypic marker with clinical course of the disease.     

Reticular fibroblasts in peripheral lymphoid organs identified by a monoclonal antibody

We have produced a panel of monoclonal antibodies directed against nonlymphoid cells in central and peripheral lymphoid organs. In this paper we present the reactivity of one of these antibodies, ER-TR7. This antibody detects reticular fibroblasts, which constitute the cellular framework of lymphoid and nonlymphoid organs and their products. In frozen sections of the spleen incubated with this antibody, the red pulp and white pulp are clearly delineated. Furthermore, the major white pulp compartments--the follicles and periarteriolar lymphoid sheath as well as the marginal zone--are recognized by their characteristic labeling patterns. In lymph nodes, the capsule, sinuses, follicles, paracortex, and medullary cords are clearly delineated. In the thymus and bone marrow no such specialized compartments were demonstrated. ER-TR7 reacts with an intracellular component of fibroblasts. Since ER-TR7 does not react with purified laminin, collagen types I-V, fibronectin, heparan sulfate proteoglycan, entactin, or nidogen, it detects a hitherto uncharacterized antigen. The possible role of the ER-TR7 positive reticular fibroblasts in the cellular organization of peripheral lymphoid organs will be discussed.     

单个B细胞抗体制备技术及应用

单克隆抗体是现代生命科学研究的重要工具,为许多领域的发展作出了不可估量的贡献。随着PCR技术和单克隆抗体技术的发展和成熟,单个B细胞抗体制备技术迅速兴起。该技术能够对单个的抗原特异性B细胞进行抗体基因的体外克隆和表达,保证了轻重链可变区的天然配对,相较于传统的抗体制备技术具有效率高、全人源、基因多样性更丰富等优势。单个B细胞抗体制备技术已成为制备全人抗体的热门方法,同时也促进了包括抗体发生成熟、疫苗保护机制、疫苗开发、肿瘤及自身免疫疾病等免疫学相关研究。文中就单个B细胞抗体制备技术的过程及应用作简要综述。     

Activation of human lymphocytes by a monoclonal antibody to B lymphoblastoid cells; molecular mass and distribution of binding protein

A novel monoclonal antibody (BAT) to the B-lymphoblastoid cell line activates murine lymphocytes and exhibits a striking antitumor activity in mice. In order to evaluate the potential use of this antibody against human cancer, we have investigated its immuno-stimulatory properties on human peripheral blood lymphocytes (PBL). Our findings demonstrate that BAT mAb induces proliferation and cytotoxicity in human PBL against natural-killer-cell-sensitive and natural-killer-cell-resistant tumor cell lines. Interleukin-2 at a low concentration synergizes with BAT mAb in eliciting these effects. BAT mAb binds to human peripheral T cells as revealed by a double-labelling technique using anti-CD3 and BAT mAb. The molecular mass of the antigen recognized by BAT mAb was 48–50 kDa under reducing and non-reducing conditions. This study provides a basis for future experiments to evaluate the use of BAT mAb in the immunotherapy of cancer.Supported by research grant (to B.H.) from the Chief Scientist, Ministry of Health, Israel     

Characterization of a family of nuclear and chromosomal proteins identified by a monoclonal antibody

A monoclonal antibody (3C5) isolated from a mouse immunized with human chromatin stained the nuclei of all cultured cell types tested by indirect immunofluorescence. Experiments with HeLa and PtK1 cells demonstrated striking cell-cycle-related changes in the staining properties of the target antigen. A rapid increase in nuclear fluorescence was seen in prophase, with antigen located between the condensing chromosomes. In metaphase and anaphase cells antigen was present throughout the cytoplasm with the chromosomes apparently unstained. However, isolated metaphase chromosomes showed intense, peripheral staining. In telophase cells immunofluorescent staining was most intense among the decondensing chromosomes and by early G1 staining was predominantly nuclear. Nuclear fluorescence faded as cells progressed through interphase. By protein blotting and immunostaining, 3C5 recognized protein bands with subunit molecular weights of 130, 73, 50, 38, 32 and 22 to 25 kDa. These bands were present in all human and rodent cultured cell types tested. All bands were extracted by 6 M urea or 1% sodium dodecyl sulfate (SDS) but not by Triton X-100. Our results provide evidence against the involvement of a common carbohydrate moiety, in vitro proteolysis or non-specific cross reaction in this multi-banded pattern. The same family of proteins was detected in mitotic and interphase cells, suggesting that the changes in immunofluorescent staining through mitosis are due to changes in antigen accessibility. Subcellular fractionation experiments showed that all major bands were present in the nuclear fraction. Only two (50 and 32 kDa) were detected also in the post-nuclear membrane fraction and none were present in the soluble cytoplasmic fraction.(ABSTRACT TRUNCATED AT 250 WORDS)     

Conformational epitope on gp120 important in CD4 binding and human immunodeficiency virus type 1 neutralization identified by a human monoclonal antibody.

A human monoclonal antibody designated 15e is reactive with the envelope glycoprotein (gp120) of multiple isolates of human immunodeficiency virus type 1 (HIV-1). Antibody 15e also neutralizes HIV-1 with broad specificity and blocks gp120 binding to CD4. Characterization of the 15e epitope shows that it is conformation dependent and is distinct from previously recognized functional domains of gp120, suggesting that this epitope represents a novel site important for HIV-1 neutralization and CD4 binding. These findings have implications for the development of a vaccine for AIDS.     

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.     

Amplification of human B cell activation by a monoclonal antibody to the B cell-specific antigen CD22, Bp 130/140

The B cell-specific antigen CD22 is a 130/140-Kd complex and is unique among human B cell antigens, since its surface expression is restricted to a subpopulation of Ig+ B cells. Here the function of the CD22 antigen was evaluated by using the mAb HD6, directed against one of the epitopes on the molecule. The HD6 antibody was constimulatory with anti-Ig in inducing small, dense tonsillar cells to proliferate; however, the antibody by itself was devoid of stimulatory activity. Anti-CD22 antibody also induced more anti-Ig-treated B cells to leave G0 and enter the G1 phase of the cell cycle. It also was constimulatory with low-m.w. BCGF and with an antibody to a 50-Kd polypeptide, Bp50, which mediates a BCGF-like activity. Results of kinetic experiments and analysis of different B cell fractions suggested that anti-CD22 acts during an early phase of B cell activation, probably by amplifying the anti-Ig signal. F(ab')2 fragments of anti-CD22 HD6 were as effective as the whole antibody in inducing augmentation of B cell proliferation, showing that the Fc portion of the molecule was not required for the activity. The results of these experiments, together with the intriguing distribution of the Bp 130/140 antigen in B cell ontogeny, suggest that this molecule plays an important role in the process that leads to B cell activation and proliferation.     

The subpopulation structure of human T-lymphocytes studied by using a monoclonal antibody (MCA) to CD27]

The obtained murine mAb LT27 (IgG2a) assigned to the cluster of differentiation CD27 was used to study the distribution of antigen CD27 among human lymphocytes scbpopulations in normal state and immunopathology. In normal donors the antigen CD27 was found to be expressed most frequently on CD4+ cells (90 +/- 8% of which coexpressed antigen CD27) and to the lesser extent on- CD8+ cells (only 77 +/- 28% of CD8+ cells carried antigen CD27). 79 +/- 12% of double negative lymphocytes (CD3+CD4-CD8-) expressed antigen CD27. In patients with hypogammaglobulinemia the proportion of CD4+CD27+/CD4+ and CD8+CD27+/CD8+ was significantly reduced to 80 +/- 11% (p < 0.01) and 45 +/- 19% (p < 0.001), respectively. The ratio CD4+CD27+/CD4+ varied insignificantly with the increase of CD4+ population, but the increase of the CD8+ population was accompanied by the definite tendency to a decrease of the ratio CD8+CD27+/CD8+. The distribution of CD27 antigen inside CD4+ and CD8+ subpopulations was found to be different from the distribution of CD29 and CD45RA antigens.     

Rendomab B4, a monoclonal antibody that discriminates the human endothelin B receptor of melanoma cells and inhibits their migration

Metastatic melanoma is an aggressive cancer with a poor prognostic, and the design of new targeted drugs to treat melanoma is a therapeutic challenge. A promising approach is to produce monoclonal antibodies (mAbs) against the endothelin B receptor (ETB), which is known to be overexpressed in melanoma and to contribute to proliferation, migration and vasculogenic mimicry associated with invasiveness of this cancer.

We previously described rendomab-B1, a mAb produced by DNA immunization. It is endowed with remarkable characteristics in term of affinity, specificity and antagonist properties against human ETB expressed by the endothelial cells, but, surprisingly, had poor affinity for ETB expressed by melanoma cells. This characteristic strongly suggested the existence of a tumor-specific ETB form. In the study reported here, we identified a new mAb, rendomab-B4, which, in contrast to rendomab-B1, binds ETB expressed on UACC-257, WM-266-4 and SLM8 melanoma cells. Moreover, after binding to UACC-257 cells, rendomab-B4 is internalized and colocalizes with the endosomal protein EEA-1. Interestingly, rendomab-B4, despite its inability to compete with endothelin binding, is able to inhibit phospholipase C pathway and migration induced by endothelin. By contrast, rendomab-B4 fails to decrease ERK1/2 phosphorylation induced by endothelin, suggesting a biased effect on ETB.

These particular properties make rendomab-B4 an interesting tool to analyze ETB-structure/function and a promising starting point for the development of new immunological tools in the field of melanoma therapeutics.     

Characteristics of the epitope of leukotriene B4 recognized by a highly specific mouse monoclonal antibody

Mouse monoclonal IgG2b antibodies to leukotriene B4 bind [3H]leukotriene B4 with an affinity one-thirtieth to one-third that of different rabbit antibodies to leukotriene B4. The concentrations of related ligands required to inhibit by 50% the binding of [3H]leukotriene B4 define cross-reactivities of approximately 100% for carboxyl-derivatives of leukotriene B4, 10% for 12(S)-leukotriene B4 and 8 cis-leukotriene B4, which were not distinguished from leukotriene B4 by polyclonal antibodies, 3-5% for the two isomers of 6 trans-leukotriene B4, 5% for 20-OH-leukotriene B4 and 20-COOH-leukotriene B4, and less than 1% for other leukotrienes, mono-hydroxy-eicosatetraenoic acids, and the two leukotriene B4-like isomers of 8, 15-di-hydroxy-eicosatetraenoic acid. Thus the monoclonal combining site is highly specific for the di-hydroxy-triene portion of leukotriene B4.     

A complex of platelet glycoproteins Ic and IIa identified by a rat monoclonal antibody

A rat monoclonal antibody, GoH3, recognizes cell surface antigens on epithelial cells in a variety of tissues in both man and mouse. Furthermore, the antibody showed reactivity with endothelial cells and blood platelets. The molecule recognized by GoH3 on platelets was determined by immunoprecipitation, followed by analysis on one- and two-dimensional sodium dodecyl sulfate-polyacrylamide gels. GoH3 precipitated glycoproteins Ic and IIa from both human and mouse platelets. Glycoprotein Ic consists of disulfide-linked heavy and light chains which both appeared to be glycosylated. As determined by enzymatic digestion followed by gel analyses, both "complex" and "high mannose" type of N-linked oligosaccharides are present on the heavy and light chain of human glycoprotein Ic and on the heavy chain of mouse glycoprotein Ic. The light chain of mouse glycoprotein Ic only carries high mannose type of N-linked oligosaccharides. The N-linked glycans on human and mouse glycoprotein IIa are all of the complex type. The glycoproteins Ic and IIa co-sedimented in sucrose gradients and formed complexes upon treatment of intact platelets with the chemical cross-linking reagent dithiobis(succinimidyl propionate). Dissociation of the complex by chaotropic agents followed by immunoprecipitation establishes that the epitope recognized by GoH3 is located on the Ic molecule. These results provide evidence that the two glycoproteins, Ic and IIa, exist as a heterodimer complex in the platelet membrane.