Abstract: Three patients (2 female, 1 male) with recurrent infection, granulocytosis, impaired pus formation, and/or delayed umbilical cord separation were identified. Assessments of polymorphonuclear leukocytes (PMN)/monocyte function in each patient revealed profound abnormalities of adherence and adherence-dependent functions. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of their PMN lysates demonstrated a deficient or absent protein(s) of 138 kilodaltons (gp 138). Na3HB4 labeling demonstrated the absence of a major cell surface glycoprotein complex in each patient. Among parental and sibling PMN suspensions, functional assessments revealed no consistent abnormalities, although variably diminished gp138 was identified by SDS-PAGE and Na3HB4 labeling. Analysis by fluorescence-activated cell sorting and monoclonal antibodies (MAb) to LFA-1 alpha, OKM1 alpha, and their common beta subunit demonstrated a severe or total deficiency of PMN/monocyte surface expression of each protein among all patients; intermediate values were observed for parental and affected sibling suspensions, findings consistent with an autosomal recessive mode of inheritance for this disorder. Cell surface labeling (125I) and immunoprecipitation with the same MAb demonstrated the absence of these glycoproteins in addition to a 150-kilodalton protein (p150,95). Identical abnormalities of surface expression of patient lymphocytes blast-transformed with phytohemagglutinin (PHA) or Epstein-Barr virus were demonstrated. Further, significantly diminished natural killer cell cytotoxicity was observed for each patient tested. PHA blast-transformed patient lymphocytes labeled with [35S]methionine demonstrated a total absence of the beta molecule but indicated the presence of an LFA-1 alpha precursor. These findings indicate that LFA-1 alpha synthesis and surface expression require beta association. It is concluded that impaired inflammatory function in this disorder is casually related to a heritable deficiency of critical "adhesive" leukocyte glycoproteins.

Abstract: Stromal cells play a critical role in haematopoiesis, both in a permissive and, probably, in a directive manner1–5. Study of the interactions between stromal cells and haematopoietic stem cells, however, is difficult to perform using whole bone marrow, in which stem cells are indistinguishable from precursor cells and maturing haematopoietic cells, and where stromal and haematopoietic cells co-exist in a heterogeneous mixture. We have purified primitive haematopoietic spleen colony-forming cells (CFU-S) using fluorescence-activated cell sorting (FACS) and produced CFU-S populations which approach 100% purity (ref. 6 and B.I.L. and E.S., in preparation). This cell population is devoid of significant stromal cells and mature haematopoietic cells. Here, we report that when purified CFU-S are seeded onto a stromal adherent layer in vitro, foci of haematopoietic cells develop within the stroma followed by production of a wave of maturing and mature progeny. However, self-renewal of CFU-S does not occur and haematopoietic activity rapidly declines, indicating that caution should be applied in the use of highly purified stem cells for human bone marrow transplantation.

Abstract: Distinct populations of human B lymphocytes can be identified by their expression and/or co-expression of the B cell-restricted antigens B1 and B2. Dual fluorochrome staining and flow cytometric cell sorting permitted the isolation of the B1+B2+ and B1+B2- cells to homogeneity. In contrast, very few B1-B2+ cells were obtainable from normal lymphoid organs. Virtually all B1+B2+ cells expressed IgM and IgD, but lacked IgG and the plasma cell antigens PCA-1 and PC-1, whereas the B1+B2- cells more frequently expressed IgG, PCA-1 and PC-1. Both populations were noncycling and were composed of similar percentages of small and large cells. The B1+B2+ cells proliferate to anti-mu or to anti-mu + PHA-LCM, but not to PHA-LCM alone. They require both T cells and PWM to produce Ig. In contrast, B1+B2-cells do not significantly proliferate to anti-mu, PHA-LCM, or anti-mu and PHA-LCM. They produce Ig in response to T cells alone without PWM. These phenotypic and functional observations provide preliminary evidence that these populations are distinct and that the B1+B2+ cell may be a "resting" B cell, whereas the B1+B2- cell appears to be more "differentiated." The present studies further suggest that they will also be helpful in characterizing B cells in some human disease states. We believe that the identification and isolation of these and similar subsets of B cells defined by differing cell surface phenotype should aid our understanding both of normal B cell differentiation and of B cell disease states.

Abstract: Cultured human epidermal cells were studied by cell sorting and autoradiography after different 3H-thymidine (3H-dThd)-labelling procedures and after labelling with DNA precursors that are incorporated via salvage or de novo pathways. It was shown that 3H-dThd incorporation was the best measure of the rate of DNA replication. Dose-response experiments with pulse and continuous labelling revealed that all S- and G2-phase cells were cycling, whereas some 20% of the cells stayed in G1-phase for long periods of time. Most, if not all of these cells were probably non-proliferating differentiated keratinocytes. At least two subpopulations of S-phase cells could be discriminated on the basis of the rate of incorporation of DNA precursors. the difference in precursor incorporation did not seem to be caused by differences in nucleotide metabolism but rather to reflect true differences in the rate of DNA replication. Continuous labelling experiments showed that these subpopulations also were apparent in the G1- and G2-phases. Studies of the grain-count distribution revealed that cells that appeared to move rapidly through the S-phase moved slowly through the G2-phase, and vice versa. Cells stained with acridine orange were subjected to a two-parameter analysis in the cell sorter by simultaneous measurement of the DNA and RNA fluorescence. Autoradiography of sorted cells revealed that, on average, cells with low RNA contents incorporated 3H-dThd at a higher rate than cells with high RNA contents.

Abstract: A mouse monoclonal antibody, WM-15, has been developed which reacts with a human myeloid lineage-restricted cell surface antigen. WM-15, an IgG1 antibody, reacts with a mean of 3.8% of normal bone marrow mononuclear cells, identifying predominantly promyelocytes and myeloblasts, and in fluorescence-activated cell sorting experiments produces enrichment of bone marrow granulocyte-macrophage progenitor cells. Normal mature monocytes and granulocytes are weakly labelled by WM-15, but other haemopoietic cells including erythroblasts and all lymphoid cells are unreactive. The myeloid specificity of this antibody is highlighted by its reactivity with myeloid leukaemia cell lines and 65% of cases of acute myeloid leukaemia, while lymphoid cell lines and leukaemias are WM-15 negative. WM-15 appears to be a useful reagent for further investigation of normal and abnormal myelopoiesis.

Abstract: Cells capable of synthesizing a factor that can specifically inhibit the proliferation of haemopoietic spleen colony-forming units, CFU-S, carry many of the characteristics of macrophages. The monoclonal antibody, F4/80, which is specific for murine macrophages, has therefore been used to isolate macrophages from bone marrow. These macrophages were then assayed for their ability to produce the inhibitor. Low-density bone marrow cells were first separated by a density-cut procedure and then labelled with F4/80. Fluorescence-activated cell sorting was then used to select F4/80 positive and negative cell fractions. It was found that the F4/80 positive fraction contains inhibitor cells, enriched by at least 200-fold compared to unfractionated marrow. Subsequent culture of this fraction over a period of several weeks produced a further 15- to 20-fold increase in inhibitor-producing capacity. The cultured cells producing inhibitor were virtually 100% F4/80 positive, phagocytic and exhibited histochemical properties characteristic of macrophages. The macrophage-like character of the producer cells was thus confirmed and the removal of the majority of unwanted cells means that many of the impurities in the normal crude extracts have been excluded.

Abstract: By use of two-parameter flow cytometry of rat testis cell suspensions stained with mithramycin for DNA (the peak amplitude of the fluorescence signal versus total fluorescence intensity integrated over time), eight cell compartments could be distinguished without pre-enrichment of the samples. Cells in these compartments were identified by sorting and subsequent microscopic examination.

Abstract: The fraction of daughter cells ofS.cerevisiae YT76 born without plasmids increases significantly during the postexponential phase of batch growth. Plasmid segregation data presented is obtained using a new method based on rapid flow cytometry-cell sorting isolation of subpopulations consisting of newborn daughter cells and dividing mother cells.

Abstract: The smooth muscle cell population in major arteries of humans and experimental animals is heterogeneous with regard to cellular DNA content. A proportion of cells has polyploid DNA content and this proportion increases with normal aging and with hypertension. We have isolated pure populations of rat aortic smooth muscle cells containing 2C, 4C, and 8C DNA content by cloning of cultures of cells previously subjected to flow cytometric cell sorting. Karyologic analysis of these clonal populations revealed them to be pure diploid, tetraploid, and octaploid populations, respectively, containing 2N (= 42), 4N, and 8N chromosomes. Cell attachment area and nuclear size appeared to increase with the level of ploidy. Studies of the proliferative characteristics of the cells revealed that the growth rate and ultimate cell densities achieved decreased as the ploidy level increased. The intrinsic cellular radiosensitivity of these clones did not vary with ploidy. Increased smooth muscle cell ploidy is, therefore, associated with a decreased rate of proliferation. The emergence of smooth muscle cells with polyploid DNA content under normal and pathologic conditions is probably due to mitotic polyploidization without net cell proliferation and may be related to the need for expression of differentiated functions.

Abstract: Cell kinetic studies on cultured human epidermal cells have indicated that cycling basal cells may be divided into at least two subpopulations that seem to differ with respect to the rate of DNA replication. The present study was undertaken in order to elucidate the biological significance of these subpopulations. The proliferation characteristics of cultured basal cells were changed by the addition of epidermal growth factor (EGF) and cholera toxin to the culture medium. It was shown that EGF and cholera toxin stimulated the growth of human epidermal cells in culture. Simultaneously, the terminal differentiation of the cells was inhibited resulting in a reduced multilayering and a reduced formation of the cornified envelope. However, only minor differences in the protein synthesis pattern were observed between cultures maintained in the presence or absence of the growth stimulators. The effect of EGF and cholera toxin on the basal cell subpopulations was investigated after3H-thymidine labelling followed by cell sorting and autoradiography. In the presence of EGF and cholera toxin dramatic changes were induced in the labelling pattern of sorted S-phase cells indicating significant alterations in the balance between the subpopulations of cycling basal cells. Our results with these substances are in accord with the hypothesis that the observed cell kinetic subpopulations may be related to regeneration or early events in the differentiation process of the keratinocyte.

Abstract: Publisher Summary This chapter focuses on fluorescence cell sorter techniques in immunology. A primary goal in the analysis of cell surface phenotype is to find markers that are correlated with a particular capability. The purpose of flow cytometry is to identify subpopulations of cells, to determine the frequency of subpopulations, and to quantify various parameters associated with the subpopulations. Flow cytometry is a method by which certain properties of cells are measured in a flow system. The flow system consists of a stream in which cells are injected and the measurements made. In such a system, many parameters can be measured, such as fluorescence, light scattering, time of flight, and Coulter volume. Cell sorting is the process of isolating cells based on parameters measured by flow cytometry. The most basic form of analysis of flow cytometric results is that of a single parameter.

Abstract: Antigenically different subpopulations of human large granular lymphocytes (LGL) were identified according to their reactivity with monoclonal antibodies (MoAb). Antigen-positive and -negative subsets were isolated by immunoaffinity columns using a Sepharose 4B gel coupled with F(a')2 goat anti-mouse IgG or by flow cytometry cell sorting. The distinct LGL subsets were tested for natural killer (NK) activity against a panel of tumor targets: K562, Daudi, Alab; and for antibody-dependent cellular cytotoxicity (ADCC) against antibody-coated RL male 1 cells. LGL positively selected for any of the following phenotypic markers: B73.1+, OKM1+, OKT11+, and OKT10+ were highly cytotoxic, while B73.1- and OKM1- cells were completely devoid of NK activity. The OKT10- and OKT11- LGL subsets were occasionally cytotoxic, with low levels of reactivity. LGL subpopulations were also tested in a limiting dilution assay (LDA) for their capacity to proliferate in medium supplemented with interleukin 2 (IL-2) and to develop NK-like cytotoxic activity. The majority of proliferative progenitors have the following phenotype: OKT11+, OKM1-, B73.1-, and OKT10-, while the majority of progenitors for cytotoxic cells were OKT11+, OKM1+/-, OKT10+, and B73.1-. Results indicate that although B73.1+ cells can grow, the mature B73.1+ NK cells seem to be primarily derived in vitro from a small subset of less differentiated B73.1 pre-NK progenitors in the peripheral blood lymphocytes.

Abstract: The gene (named MFI6) for a surface membrane antigen, Trop-4, is assigned to human chromosome 11 on the basis of studies using a mouse monoclonal antibody, immunofluorescence, fluorescence-activated cell sorting (FACS), immunoprecipitation, and mouse-human lymphocyte hybrids. The Trop-4 antigen is present on all human cell lines tested, on peripheral blood monocytes and granulocytes, and on a small fraction of peripheral blood lymphocytes, but is absent from erythrocytes. The Trop-4 monoclonal antibody precipitates an 85,000-dalton glycopolypeptide from hybrid cells containing human chromosome 11. However, in a human cell line expressing this antigen, a larger-molecular-weight species, 100–105,000 daltons was coprecipitated with the 85,000-dalton glycopeptide, and under nonreducing conditions a larger compound of 110–125,000 daltons was obtained. Although the Trop-4 antigen is of similar molecular weight to the Mab-4 and F10.44.2 antigens previously assigned to chromosome 11, it is shown to be different from them.

Abstract: In this report we describe some properties and applications of two different groups of monoclonal antibodies (Mab) produced in our laboratory for the analysis of molecular and cellular aspects of the process of carcinogenesis induced by N-ethyl-N-nitrosourea (EtNU) in the developing rat brain, Mab of the first group are directed against defined alkylation products in the DNA of cells exposed to alkylating N-nitroso compounds. In conjunction with sensitive immunoanalytical methods, these Mab are used for quantitation of alkyl-deoxynucleosides in the DNA of tissues and individual cells, for studies of DNA repair, and for analyses of the frequency and distribution of specific alkyldeoxynucleosides in the DNA of different chromatin fractions and in individual DNA molecules. Mab of the second group recognize and bind to cell type- and developmental stage-specific cell surface determinants (CSD) of rat brain cells (BC). These Mab are presently applied for the separation by fluorescence-activated cell sorting (and subsequent in vitro culture) of distinct BC subpopulaticns previously exposed to EtNU in vivo, as well as for comparative CSD analyses of defined subpopulations of BC and their malignant counterparts.

Abstract: Expression of developmentally regulated membrane proteins of aggregating cells of Dictyostelium discoideum is subject to several control mechanisms. One of them involves periodic cyclic-AMP pulses as signals for gene expression. To increase the probability of selecting mutants specifically defective in the contact site A (csA) glycoprotein, one of the characteristic proteins of aggregating cells, we have bypassed the requirement for both cyclic-AMP pulses and another control element by two runs of mutagenesis. A ;double bypass' mutant, HG592, was obtained which aggregated in nutrient medium where wild-type did not develop. Mutants defective in expression of the csA-glycoprotein were selected from HG592 by fluorescence-activated cell sorting and colony immunoblotting using a monoclonal antibody specific for that protein. One among 51 csA-negative mutants, HG693, specifically lacked the capability of forming EDTA-stable intercellular contacts. It acquired chemotactic responsiveness and developed into fruiting bodies. Expression of the transcripts for eight developmentally regulated proteins was determined in HG693. Seven of the RNA species were normally expressed; they were recognized by cDNA clones which had been produced from poly(A) RNA isolated from membrane-bound polysomes. The single RNA species which was not substantially expressed in HG693 was recognized by a cDNA clone that was obtained by screening a lambdagt11 library with an antibody specific for the csA-glycoprotein. When probing RNA from wild-type cells, this clone hybridized with a single developmentally regulated RNA species of 1.9 kb whose expression was strongly enhanced by cyclic-AMP pulses. Appearance of this RNA coincided with the expression of the csA-glycoprotein.

Abstract: DNA from the human myeloid cell line HL-60 was cotransfected with the cloned thymidine kinase (tk) gene of herpes simplex virus into tk-deficient mouse L cells. tk-positive recipients expressing antigens detected on HL-60 cells were isolated with a fluorescence-activated cell sorter by use of a panel of monoclonal antibodies that detect epitopes on both normal and malignant myeloid cells. Independently sorted populations of transformed mouse cells showed concordant reactivities with four of the monoclonal antibodies in the panel (DU-HL60-4, MY7, MCS.2, and SJ-D1), which suggested that these antibodies reacted to products of a single human gene. A second round of DNA transfection and cell sorting was performed with donor DNA from primary transformants. Two different dominant selection systems were used to isolate secondary mouse L cell and NIH/3T3 cell transformants that coexpressed the same epitopes. Analysis of cellular DNA from secondary mouse cell subclones with a probe specific for human repetitive DNA sequences revealed a minimal human DNA complement containing a characteristic set of restriction fragments common to independently derived subclones. Two glycoproteins, of 130,000 (gp130) and 150,000 (gp150) mol wt, were specifically immunoprecipitated from metabolically labeled lysates of mouse cell transformants and were shown to contain [35S]methionine-labeled tryptic peptides identical to those of analogous glycoproteins expressed in the donor human myeloid cell line. Kinetic and biochemical analyses established that gp130 is a precursor that differs in its carbohydrate moiety from gp150, the mature form of the glycoprotein detected on the cell surface. The isolation of human gene sequences encoding gp150 in a mouse cell genetic background provides the possibility of molecularly cloning the gene and represents a general strategy for isolating human genes encoding differentiation-specific cell surface antigens.