Membrane immunoglobulin without sheath or anchor.

The canonical form of the B cell antigen receptor is composed of membrane immunoglobulin sheathed by the alpha/beta heterodimer. Whereas membrane IgM cannot be transported to the cell surface in the absence of alpha/beta, both IgD and IgG2b can be expressed naked (i.e. without alpha/beta) on the surface of myeloma transfectants. In the case of one cell-line, such naked IgD has been shown to be inserted into the membrane by a glycosyl-phosphatidylinositol anchor. Here, however, we show that both IgD and IgG2b (but not IgM) can be expressed on the surface of myeloma transfectants without either sheath or anchor. This distinction between the isotypes is attributable to differences in the region of the transmembrane segment.

CD66 identifies a neutrophil-specific epitope within the hematopoietic system that is expressed by members of the carcinoembryonic antigen family of adhesion molecules.

Preliminary results from the IVth Leucocyte Culture Conference have classified the monoclonal antibody (MoAb), YTH 71.3.2, as CD66. Two other MoAbs, YPC 2/12.1 and CE6/2D3.1, share a common cellular specificity, reacting with cells of the neutrophil series and colonic epithelium. The YTH 71.3.2 and CE6/2D3.1 MoAbs both recognize a similar CD66 defined epitope that is distinct from that identified by YPC 2/12.1. By Western blotting, these antibodies react with different molecular species from cells of different lineages. The antibodies identify 50- to 55-Kd, 80- to 100-Kd, and 130- to 200-Kd components present in a semi-purified carcinoembryonic antigen (CEA) preparation from colonic adenocarcinomas and a 90- to 130-Kd molecule from HL-60 cells. With the colonic cell line, LS174T, YPC2/12.1 stains diffuse bands of 160 to 200 Kd and 90 to 130 Kd with equal intensity, whereas the binding of CE6/2D3.1 and YTH 71.3.2 is biased toward the lower molecular weight set of molecules. Remarkably, all three antibodies recognize CEA-related molecules. Defined analyses using HeLa cells transfected with CEA, NCA(NCA-50/90), and CGM6(NCA-95) cDNAs show that the three MoAbs identify CEA to varying degrees. While YTH 71.3.2 and CE6/2D3.1 also bind to NCA-50/90, YPC 2/12.1 recognizes an epitope expressed by both the NCA-50/90 and NCA-95 molecular species.

The sequence of the mu transmembrane segment determines the tissue specificity of the transport of immunoglobulin M to the cell surface.

Membrane IgM is expressed on the surface of B lymphocytes. It is not transported to the surface of transfected plasmacytoma or COS cells. Here, we show that mutation of four hydrophilic amino acids in the microm transmembrane is sufficient to overcome the intracellular retention of membrane IgM in non-B cells. This suggests that the B cell-specific IgM-associated proteins that have been postulated to assist the transport of membrane IgM to the cell surface (3) act either by forming a hydrophobic sheath that surrounds the microm transmembrane segment or by displacing an interaction with this segment that would otherwise cause retention. Experiments with a CD8/mu hybrid H chain indicate that the proteins that assist the transport of membrane IgM to the B cell surface at most need the mu CH4 and transmembrane/cytoplasmic portion for interaction.

Phorbol esters potentiate the induction of class I HLA expression by interferon alpha.

We have studied the effect of phorbol esters on the induction of class I histocompatibility antigen (HLA) expression by interferons (IFNs) in the T-cell line MOLT-4 and in the MOLT-4 mutant YHHH. Addition of IFN-alpha to phorbol 12,13-dibutyrate-pretreated MOLT-4 cells causes a greater than 20-fold increase in the expression of class I HLA, as compared to a 4- to 7-fold IFN-alpha-induced increase in control cells. Pretreatment with phorbol 12,13-dibutyrate does not alter the class I HLA response to IFN-gamma or the responses of other IFN-induced genes. This effect of phorbol 12,13-dibutyrate reproduces in MOLT-4 cells the phenotype of the mutant YHHH, which also displays a selective enhanced class I HLA response to IFN-alpha. Pretreatment of YHHH with phorbol 12,13-dibutyrate does not affect any of the responses induced by IFN. These findings suggest the existence of a phorbol ester-sensitive factor, inducible in MOLT-4 and constitutively expressed or modified in YHHH, which operates in the pathway of induction of class I HLA by IFN-alpha but not in the pathway used by IFN-gamma.

Cell-surface markers on haemopoietic precursors. Reagents for the isolation and analysis of progenitor cell subpopulations.

Within the last decade, major advances have been made in the analysis of cell-surface marker expression on haemopoietic progenitor cells as a result of the development of multiparameter cell sorting and monoclonal antibody techniques. Although some controversy exists with regard to the actual identification of the stem cell, markers specific for CFU-s and for particular subsets of progenitor cells have not yet been identified. An analysis of cell-surface markers on haemopoietic progenitor cells is complicated by at least three factors. First, it appears that, in mice, the clonal assays do not adequately identify the haemopoietic stem cell. Complete repopulation of all haemopoietic cell compartments in vivo over an extended period of time appears to be the only reliable method for identifying such a cell. Secondly cell-surface marker distribution on haemopoietic progenitors from normal tissues may be indicative of the cycling status of cells. Thus, expression of markers on progenitors from bone marrow or foetal liver which have been perturbed by drugs or viruses may merely reflect a change in their cycling status following drug or viral insult. Thirdly, substantial loss of cells occurs during the purification of particular cell types. For most cell separation procedures, only a minor proportion of the progenitor cells of interest are recovered and these may not be representative of the progenitor population as a whole. During differentiation to mature cells, antigenic determinants present on early progenitor cells may either be progressively lost or amplified. This differential expression of cell-surface molecules has provided a useful tool for the substantial enrichment of haemopoietic subsets, particularly CFU-E and CFU-s. To date, however, most early haemopoietic progenitor cells detected by in vitro CFC assays (day 8 CFC) cannot be completely segregated from one another. The ability to distinguish between such progenitors during the early stages of lineage commitment would provide a more detailed understanding of the relationship between lymphoid precursors, myeloid precursors and stem cells, and would lead to significant advances in developmental biology. Separation of cells at different stages of differentiation within a given lineage would provide an opportunity for studying regulatory mechanisms involved in gene expression in normal cell populations.

Haemopoietic progenitor cell heterogeneity revealed by a single monoclonal antibody, YW 13.1.1.

Rapid segregation and purification of haemopoietic progenitor cells by simple methods is necessary for a proper analysis of the control of early haemopoiesis. In this paper we describe the use of a rat monoclonal antibody, YW 13.1.1, for that purpose. This reagent reacts with more than 90% of foetal liver cells but spares stem cells. A single-step lysis with antibody and complement achieves a tenfold enrichment for early progenitor cells. The marker also shows an increasing level of expression on the three defined subsets of erythroid progenitor cells. This parallels their developmental pathway and erythropoietin responsiveness. Simple quantitative considerations therefore permit separation of cells at different stages of erythropoiesis.

Selective isolation of murine erythropoietin-responsive progenitor cells (CFU-E) with monoclonal antibodies.

Erythropoietin-responsive progenitor cells (CFU-E) from normal mouse foetal liver have been substantially purified on the basis of their differential binding to two monoclonal antibodies in conjunction with flow cytometry. This has allowed the formal identification of the foetal liver CFU-E as an early erythroid blast cell with a highly basophilic cytoplasm. It has also been possible to show a quantitative association of a membrane marker with proliferative potential within a single differentiation lineage. This is the first demonstration of such an association. Progenitor cells enriched in this way should allow study of the molecular mechanisms of erythropoietin action and also serve as a target for determining the cellular specificity of erythroid-transforming viruses.

The characterisation of monoclonal antibodies against haemopoietic cells: comparison of an immunoperoxidase method with fluorescence activated cell sorting.

Nucleated cells from normal human peripheral blood and bone marrow have been analysed with 2 rat monoclonal antibodies of known specificity, one (YAML 555.6.6) directed against the P28,33 complex (anti-HLA-DR), and the other (YAML 501.4.4) directed against leucocyte common antigen (anti-LCA). The patterns of reactivity with an indirect immunoperoxidase method on fixed smeared cells were in close agreement with those obtained with a fluorescence activated cell sorter. A further new monoclonal antibody of unknown antigen specificity (YAML 537.2) reacts with an intracellular antigen present in neutrophils and their precursors from the promyelocyte stage onwards, megakaryocytes, and a proportion of monocytes, but not with eosinophils, nucleated red cells or lymphocytes. This reactivity could not be demonstrated using the fluorescence activated cell sorter. The immunoperoxidase method allows the identification of individual positive and negative cells and therefore provides a method of identifying minor reactive and non-reactive cell populations in a heterogeneous cell sample such as normal bone marrow. Cytoplasmic binding sites can be differentiated from membrane binding.

A neutron television camera detector.

The system under development has a large counting rate capability; this is extremely important where the total background count exceeds the total counts in the signals of interest. Its spatial resolution is of the order of one mm, which is perfectly adequate for neutron work, while the screen size of 400 mm is reasonable. The main limitation of the system is its limited counting efficiency, and this is directly attributable to the optical self-absorption of the neutron phosphor. Any newly developed transparent phosphor with the same light output would immediately change the situation. The success of the electronics hardware in reducing random noise is demonstrated in Figure 3, which shows in the bottom trace the live video output when the input to the system is a grey-scale test chart. The top trace is the output after the image has been digitally integrated. Figures 4 and 5 show the monitor outputs of the see articles x-ray system with a "still" diffraction pattern of a crystal of GPD (glyceraldehyde-3-phosphate dehydrogenase). Figure 4 is a photograph of the "live" video display, and Figure 5 is the digitally summed image. All coherent noise in the system, i.e., all noise synchronized with the TV scans has to be kept lower than the first bit threshold. However, this requirement can be relaxed when dealing with diffraction patterns, such as those from single crystals, for which a local background is subtracted from the pattern.