Delineation of human cord blood hematopoietic progenitor cell subsets with a unique monoclonal antibody AY19.

AY19, a unique mAb was used to better characterize the umbilical cord blood hematopoietic progenitor subpopulations. This mAb identifies an 85 kDa cell surface glycoprotein. In this present study we showed that AY19 mAb is reactive with 50-60% of the CD34+ cord blood cells. Extensive phenotypical studies revealed that AY19 mAb defines a novel CD34+ subset different from the ones defined by anti-CD90, anti-CD38, anti-CD33, anti-CD71, anti-CD19, anti-CD7 or anti-HLA-DR mAbs. We show that AY19 mAb reacts with both primitive and committed progenitors including myeloid and lymphoid progenitors. In addition, sorted CD34+high/AY19+ cells contain an increased number of CFU-GM and a decreased number of BFU-E compared with sorted CD34+high/AY19- cells. We show that AY19 mAb exhibits agonistic properties by inducing a significant increase in the size of CFU-GM colonies when added to serum-free liquid cultures of hematopoietic progenitors. This suggests that AY19 mAb identifies a cell surface receptor which may be involved in the regulation of hematopoietic cell proliferation.

Functional role of PECAM-1/CD31 molecule expressed on human cord blood progenitors.

CD31/PECAM-1 (platelet endothelial cell adhesion molecule-1) is a 130 kDa integral membrane protein of the immunoglobulin gene superfamily with the distinctive feature of being expressed on several cell types associated with the vascular compartment. In the present study we report a novel, unique CD31 mAb termed IP28A which reacts with all CD34 molecule expressing hematopoietic progenitor cells and a subset of T, B and NK lymphocytes from human cord blood. Interestingly, we show that the number of CFU-GM and BFU-E was significantly augmented in cord blood progenitor cultures when purified IP28A mAb was added to rhSCF plus rhGM-CSF and rhEpo, respectively. Thus, these results are of relevance in the field of hematopoietic stem cell transplantation as they reveal an agonistic property of the IP28A/CD31 mAb on the differentiation of cord blood progenitor cells.

Detection of membrane-bound HLA-G translated products with a specific monoclonal antibody.

A monomorphic anti-HLA-G monoclonal antibody (mAb) was obtained by immunization of HLA-B27/human beta 2-microglobulin double-transgenic mice with transfected murine L cells expressing both HLA-G and human beta 2-microglobulin. This mAb, designated BFL.1, specifically recognizes, by flow cytometry analysis, the immunizing HLA-G-expressing cells, whereas it does not bind to parental untransfected or to HLA-B7- and HLA-A3-transfected L cells, suggesting that it distinguishes between classical HLA-A and -B and nonclassical HLA-G class I molecules. This was further assessed by the absence of BFL.1 reactivity with a number of human cell lines known to express classical HLA class I proteins. In addition, we showed that the BFL.1 mAb also labels HLA-G-naturally-expressing JEG-3 and HLA-G-transfected JAR human choriocarcinoma cell lines as well as a subpopulation of first-trimester placental cytotrophoblast cells. Further biochemical studies were performed by immunoprecipitation of biotinylated membrane lysates: BFL.1, like the monomorphic W6/32 mAb, immunoprecipitated a 39-kDa protein in HLA-G-expressing cell lines, a size corresponding to the predicted full-length HLA-G1 isoform. However, in contrast to W6/32, which immunoprecipitates both classical and nonclassical HLA class I heavy chains, BFL.1 mAb does not recognize the class Ia products. Such a mAb should be a useful tool for analysis of HLA-G protein expression in various normal and pathological human tissues and for determination of the function(s) of translated HLA-G products.

BY55 monoclonal antibody delineates within human cord blood and bone marrow lymphocytes distinct cell subsets mediating cytotoxic activity.

We had previously reported, using BY55 monoclonal antibody, a cell surface 80-kDa protein restricted to human functional peripheral blood cytotoxic lymphocytes with either natural killer CD3- or cytotoxic T lymphocyte CD3+CD8+ phenotype. In the present report, we studied the cytotoxic lymphocytes in adult bone marrow and newborn cord blood as these organs are commonly used as sources of hematological stem cells for allogeneic transplantation. Our results showed that BY55 mAb labeled only 5-10% of the bone marrow lymphocytes, which included a major proportion of CD3+ CD8+ cytotoxic T lymphocytes. Interestingly, within cord blood cells, BY55+ lymphocytes represented 20-35% of the lymphocytes corresponding exclusively to a CD3- cell subset. Furthermore, we detected in cord blood no cytotoxic T lymphocyte activity but we demonstrated that the CD3-BY55+ cell subset contained the whole natural killer activity.

A novel 80-kD cell surface structure identifies human circulating lymphocytes with natural killer activity.

Human lymphocytes with natural killer (NK) activity, including most activated gamma/delta+ T lymphocytes, recognize and lyse tumor target cells without requiring recognition of major histocompatibility complex antigen. However, unlike gamma/delta+ T lymphocytes, NK cells do not express CD3/T cell receptor (TCR) molecules, and the receptors involved in cell-mediated cytotoxicity are unknown. To further delineate circulating NK cells, we developed monoclonal antibodies (mAbs) against the human NK leukemia YT2C2. We report the isolation of a mAb termed BY55, recognizing at the cell surface a novel 80-kD protein with restricted expression. In addition to the immunizing cell line, this mAb binds to circulating NK cells, gamma/delta+ cells, and a minor subset of alpha/beta+ T lymphocytes. Expression of the BY55 mAb-reactive epitope/molecule is regulated by activation, as short-term culture of peripheral blood lymphocytes (PBL) with phorbol ester induced its downmodulation. Furthermore, BY55 mAb reactivity was found neither with the NK nor with the TCR alpha/beta+ gamma/delta+ clones tested. Biochemical studies as well as phenotypic analysis revealed that this structure is different from all previously identified molecules on the lymphocyte cell surface. Interestingly, we found that BY55+ cells exert most NK activity obtained with fresh circulating lymphocytes. We report that within fresh E rosette-positive PBL only a subset of the CD16+, CD56+, and CD57+ cells coexpressed BY55 molecule, indicating that BY55 mAb defines a unique subset mediating NK activity of circulating PBL.

Identification of a novel functional 85-kD glycoprotein restricted to long-term dividing human lymphocytic lines.

Lymphocyte activation induces or increases the expression of several surface structures, some of which are directly involved in cell growth such as receptors for IL2 or transferrin. To identify new structures characteristic of activated lymphocytes, we developed a series of mAbs against functionally defined human T-cell clones or the NK-mediating cell line YT2C2. In this study, we report the isolation of an mAb termed AY19 recognizing, at the cell surface, an 85-kD glycoprotein whose expression is restricted to T-lymphocyte clones, leukemic T-cell lines, and T-ALL peripheral blood cells. Biochemical studies, as well as phenotypic analysis, revealed that this structure is different from all previously identified molecules on the cell surface of lymphocytes. Furthermore, functional studies showed that triggering of this 85-kD structure on cloned T-lymphocytes through the AY19 epitope led to an inhibition of CD3-induced proliferation. These findings suggest that the AY19 mAb defines a T-cell antigen expressed mainly on long-term dividing lymphocytes and, therefore, its putative ligand might play a role in the regulation of T-lymphocyte growth induced by CD3-TcR stimulation.

Increased surface expression of a newly identified 150-kDa dimer early after human T lymphocyte activation.

Lymphocyte activation induces or increases the expression of several surface structures, some of which are directly involved in cell growth such as receptors for IL-2 or transferrin. In order to identify new structures characteristic of activated lymphocytes, we developed a series of mAb against functionally defined human T cell clones. In the present study we report the isolation of a mAb termed BB18 recognizing, at the cell surface, a novel 150-kDa glycoprotein dimer whose expression on T lymphocytes increases readily after their activation with various stimuli including lectins. In contrast, in the presence of PMA, cell-surface expression of this 150-kDa structure is down-regulated even earlier than CD3 molecules. Biochemical studies as well as phenotypic analysis revealed that this structure is different from all previously identified molecules on the lymphocyte cell surface. Furthermore, functional studies showed that triggering this disulfide-linked dimer through BB18 epitope in the presence of submitogenic concentrations of PMA induced strong lymphocyte proliferation. This proliferative response require E+ cells and accessory cells, and this even after immobilization of BB18 mAb.