Surface Of Hemopoietic Cells Research Articles

Key role played by a butyrophilin family gene product in human gd T cell activation (66.39)

Abstract The major peripheral Vg9Vd2 T cell subset in humans is activated by small phosphorylated metabolites of the isoprenoid synthesis pathway (referred to as phosphoantigens (P-Ag)), which are overexpressed in tumor and infected cells. The molecular partners and the mechanisms involved in P-Ag-mediated activation of Vg9Vd2 T cells remain elusive. In line with the recently described implication of murine Skint-1, a butyrophilin (BTN) family-related molecule, in selection of murine gd T cells, we show that several mAbs against a human BTN-related product with yet unknown function, which is ubiquitously expressed at the surface of hemopoietic cells sensitize a broad array of target cells to specific Vg9Vd2 T cell recognition (cytolysis, cytokines release, proliferation). Although this process is Vg9Vd2 TCR-dependent and involves gamma/delta chain residues previously shown to be critical for P-Ag-induced activation, we failed to detect any significant modulation of MVA pathway genes expression, nor any increase of endogenous P-Ag (IPP/ApppI) accumulation within mAb-treated target cells. We also identified a BTN-specific mAb that specifically inhibited Vg9Vd2 T cell activation triggered by endogenous or exogenous P-Ag.Moreover BNT knockdown abrogated Vg9Vd2 responses to PAg. Taken together, our results support a unique and critical contribution of BTN-related products in the regulation of TCR-mediated activation of human Vg9Vd2 T cells.

Human anti-CXCR4 antibodies undergo VH replacement, exhibit functional V-region sulfation, and define CXCR4 antigenic heterogeneity.

The chemokine receptor CXCR4 and its ligand stromal-derived factor-1 (SDF-1/CXCL12) are essential for many biological processes and various pathological conditions. However, the relationship between CXCR4 antigenic structure and SDF-1-mediated biological responses is poorly understood. In this report, a panel of human anti-CXCR4 Abs were isolated and used to explore CXCR4 antigenic heterogeneity and function. Multiple fixed CXCR4 antigenic isoforms were detected on the surface of hemopoietic cells. Epitope mapping studies demonstrated the complex nature of the surface-exposed CXCR4 epitopes. Ab-mediated inhibition of chemotaxis correlated strongly with binding affinity, epitope recognition, as well as the level of CXCR4 isoform expression. In addition, detailed genetic analyses of these Abs showed evidence of V(H) replacement. Importantly, structural and biochemical studies demonstrated tyrosine sulfation in novel regions of the V genes that contributed bidirectionally to the binding activity of the Abs. These data provide the first evidence that functional tyrosine sulfation occurs in self-reactive Abs and suggest a potential new mechanism that may contribute to the pathogenesis of Ab-mediated autoimmune disease. These Abs also provide valuable tools to explore the selective in vivo targeting of CXCR4 isoforms that may be preferentially expressed in certain disease states and involved in steady-state CXCR4-SDF-1 homeostasis.

Membrane antigen expression during hemopoietic differentiation

The pattern of certain groups of antigens expressed on the surface of hemopoietic cells changes either during the course of differentiation from pluripotent stem cells to mature functional cells or as a function of the proliferative state of the cells. A map of these changes is emerging and is providing valuable information for selecting and purifying rare stem cells and for classifying the acute leukemias. This knowledge is also beginning to provide insights into physiological and pathological cellular interactions affecting the early stages of hemopoiesis, and is being exploited to remove T lymphocytes from allogeneic bone marrow grafts in order to prevent graft-vs.-host disease as well as leukemic cells from bone marrow before autologous reinfusion. In this article I will briefly review the cellular basis of hemopoiesis and then discuss the methods used to determine the presence of antigens on normal hemopoietic cells. I will then summarize the pattern of membrane antigens expressed during differentiation and conclude by discussing the biological and therapeutic implications.

Differential expression of lectin receptors during hemopoietic differentiation: enrichment for granulocyte-macrophage progenitor cells.

Molecular changes occur at the surface of hemopoietic cells during differentiation from progenitor cells to mature granulocytes and macrophages. The differential expression of surface carbohydrate residues has been probed using lectins and the results used to purify normal mouse granulocyte-macrophage progenitor cells. Ten different lectins were screened for selective interaction with mouse hemopoietic colony-forming cells (CFCs), using agglutination or a quantitative analysis of the number of fluoresceinated lectin molecules bound per cell using a fluorescence activated cell sorter (FACS). Pokeweed mitogen (PWM), Helix pomatia agglutinin (HPA), soybean agglutinin (SBA), and peanut agglutinin (PNA) preferentially bound to CFCs so that it was possible to enrich 4 to 10-fold for these progenitor cells by sorting for the highly fluorescent cells. Further analysis of the low and high angle light scattering characteristics of the CFCs indicated that these cells were polydisperse, but could be enriched ten-fold by selecting for cells with high intensity low angle (0 degrees) scatter and low intensity high angle (90 degrees) scatter. PWM gave the best enrichment (10 to 15-fold) for adult bone marrow CFCs, for CFCs from fetal sources (fetal liver, fetal blood), and for CFCs from the spleens of mice injected previously with outer membrane lipoprotein from E. coli. Three parameter sorting for CFC using the FACS (low angle scatter, high angle scatter, and PWM-fluorescence) resulted in large enrichment factors (16 to 50-fold) for CFCs from all the above sources. Over 7% of the cells sorted from bone marrow, 10% of the cells sorted from post-lipoprotein spleen, and 28% of the cells sorted from fetal peripheral blood were hemopoietic CFCs. Ninety percent of the cells in these fractions had the morphology of blast cells or myelocytes. Thus, it was possible to identify the morphological characteristics of the hemopoietic progenitor cells. Screening of other developmental systems using quantitation of fluorescence with lectins should prove of general value for the purification of selected differentiation states.

Ultrastructural localization of concanavalin A binding sites on the surface of differentiating hemopoietic cells.

The surface characteristics of hemopoietic cells in normal human bone marrow have been explored by the use of the ultrastructural concanavalin A-peroxidase-diaminobenzidine (CAPD) procedure for the detection of specific carbohydrate residues (α-D-mannopyranoside, α-D-glucopyranoside, and α-N-acetyl-D-glycosaminide) associated with the cell surface. All cells present in the bone marrow were capable of binding Con A to their surfaces. The extent of binding proved cell specific and could be related to the stage of morphological development of each cell line. Maximum surface reactivity in the bone marrow cell population occurred with the most immature cells (myeloblast and erythroblast), lymphocytes, eosinophils, monocytes, macrophages and platelets, while mature neutrophils, basophils, and erythrocytes showed only minimum surface reactivity. These findings serve not only to expand our knowledge of the chemical nature of the surface of differentiating hemopoietic cells but provide striking evidence that modifications in the surface of hemopoietic cells occur during the process of normal cell differentiation in the bone marrow.