Improved sensitivity in comparative genomic hybridization analysis of DNA heteroploid cell mixtures after pre-enrichment of subpopulations by fluorescence activated cell sorting.

Cytogenetic analysis of solid tumors with comparative genomic hybridization (CGH) is hampered by the dilution of DNA from individual tumor subpopulations with DNA from other cells. We investigated to what extent this dilution effect can be alleviated using fluorescence activated cell sorting (flow sorting) of experimental DNA heteroploid cell mixtures prior to CGH. From mixtures of normal lymphocytes with triploid K-562 cells the individual components were sorted according to stemline DNA content and processed by CGH in comparison with pure K-562 samples and the original mixtures. Compared with 30 autosome copy number imbalances found in pure K-562 samples, a mixture with 32% K-562 cells showed 16 imbalances, and none were detected in mixtures with 13% or 5% K-562 cells. In contrast, 29, 22 and 23 imbalances were detected in K-562 nuclei sorted from the 32%, 13% and 5% mixtures, respectively. This indicate that CGH analysis of flow sorted DNA aneuploid subpopulations enables a specific cytogenetic analysis of the individual subclones in a DNA heteroploid cell population.     

Cytotoxicity from cultured cells: analysis of precursors involved in generation of human cells mediating natural and antibody-dependent cell-mediated cytotoxicity.

Natural cell-mediated cytotoxicity of human peripheral blood lymphocytes natural killer (NK) against K-562 and antibody-dependent cellular cytotoxicity (ADCC) against Chang cells, as measured in a 4-hr ⁵¹Cr release assay, were both completely removed by depletion of Fc receptor-positive (FcR+) cells. After in vitro culture for 7 days, however, NK- and ADCC-like activities spontaneously regenerated. The nature of precursor cells was studied by examination of lymphocyte subpopulations required for generation of this cytotoxicity. After depletion of FcR+ cells from PBL, the following subpopulations were prepared: sheep erythrocyte rosette-forming cells (E+), surface membrane immunoglobulin-positive cells (SmIg+), and null cells (lacking E+, SmIg+, or FcR+ markers). Separate cell types or mixtures were cultured in vitro in medium containing 10% fetal calf serum for 7 days and then tested for NK and ADCC. Whereas unseparated FcR-depleted cells developed substantial cytotoxic activity, each of the subpopulations cultured alone was negative or had low activity. Addition of SmIg+ cells to other cell types had no effect; however, mixture of 80% E+ and 20% null cells resulted in optimal NK and ADCC. It is not presently clear which population the precursors were in. However, the requirement for proliferation by the null cell population but not by the E+ cells (as indicated by sensitivity to radiation and mitomycin C) suggested that the precursors for NK cells may be null cells.     

Natural cytotoxic reactivity of human lymphocytes against a myeloid cell line: characterization of effector cells.

Using a series of techniques to identify and deplete various peripheral blood lymphocyte subpopulations, we studied the cytotoxic reactivity of normal individuals against the myeloid cell line K-562 in a 4-hr 51chromium-release assay. Depletion of lymphocytes bearing complement receptors had a variable, usually negligible effect on cytotoxicity. In contrast, depletion of lymphocytes bearing Fc receptors abrogated target cell lysis. Separation of lymphocytes with high-affinity binding of sheep red blood cells (SRBC) evidenced by rosette formation at 29 degrees C yielded a population of rosette-forming cells containing few cytotoxic cells, whereas separation of total E-RFC under optimal rosetting conditions produced a rosette fraction containing a major proportion of the effector cells. These data indicate that the cytotoxic lymphocyte in this system is Fc receptor positive, largely complement receptor negative, and may possess low density or low affinity receptors for SRBC.     

Distinct subpopulations of IgG memory B cells respond to different molecular forms of the same hapten.

Spleen cells from mice primed with the thymus-dependent antigen trinitrophenyl keyhold limpet hemocyanin several months earlier can be cultured in vitro to give vigorous IgG antihapten PFC responses to thymus-dependent (TD) and thymus-independent (TI) forms of the same hapten. Here we show that the IgG memory precursors that respond to these two forms of the hapten constitute functionally distinct subpopulations. We have designated these subpopulations as B1gamma and B2gamma to represent secondary precursor cells responding to TI and TD antigens, respectively. Three types of evidence for these subpopulations are presented: 1) In vitro secondary IgG responses to TD and TI forms of the TNP hapten are additive when both forms are added to the same culture. 2) The precursor frequencies for the TD and TI antigens are additive, but addition is not observed between two TD or two TI antigens. 3) Each population can be selectively eliminated by BUdR and light treatment without affecting the other population. The ontogenetic relationships between these subpopulations are discussed in relation to all presently proposed subpopulations B1mu, B2mu, B1gamma, and B2gamma.     

Glucocorticoid receptors in subpopulations of human lymphocytes defined by monoclonal antibodies

Glucocorticoid receptors (GR) were investigated in subpopulations of lymphocytes identified by monoclonal antibodies. Purified T (OKT3+) and non-T lymphocyte subpopulations were isolated from human peripheral blood using Degalan bead columns coated with rabbit anti-human IgG. Purified subpopulations of OKT4+ and OKT8+ lymphocytes were obtained by coating the nonadherent population (T cells) from the first column with OKT4+ or OKT8+ and pouring it into a second Degalan column, coated with goat anti-mouse IgG. GR content and affinity were analyzed by a whole cell assay with [3H]dexamethasone as tracer. The numbers of GR in lymphocyte subpopulations (OKT3+ cells, non-T cells, OKT4+, and OKT8+ cells) were nearly equal. It is concluded that the differential effects of glucocorticoids on the circulatory kinetics of OKT4+ and OKT8+ cells probably are not related to differences in glucocorticoid receptors of these T-cell subpopulations.     

Subpopulation analysis of human granular lymphocytes: associations with age, gender and cytotoxic activity

Levels of human blood granular lymphocyte subpopulations were enumerated in 105 healthy donors ranging in age from 0 to 79 years. Using double-label immunofluorescence, subpopulations of granular lymphocytes were enumerated as follows: Leu7+Leu2+, Leu7+Leu3+, Leu7+11-, Leu7+77+ and Leu7-Leu11+. The proportion of cells with granular lymphocyte morphology was determined by Giemsa staining. Natural killer (NK) cell activity against K-562 cells and lymphokine-activated killer activity against non-cultured melanoma cells were examined in parallel. Levels of total Leu7+ and Leu11+ cells increased with age (p = 0.0001) and were higher in males than females (p = 0.001). The total number of cells with granular lymphocyte morphology had an age-related increase (p = 0.001), but were not significantly higher in males than in females (p = 0.07). There was no selective increase in one granular lymphocyte subpopulation versus another since the Leu7+Leu11- (p = 0.0001), the Leu7+Leu11+ (p = 0.0001), the Leu7+Leu2+ (p = 0.0001) and the Leu7+Leu3+ (p = 0.0004) all had similar age-related increases. The one exception was the Leu7-Leu11+ (p = 0.1) granular lymphocyte subset which was low in the first decade of life but had reached maximum levels in the second decade. NK cell activity against K-562 cells was moderately increased with age (p = 0.06) with males and females exhibiting comparable activity. In contrast. lymphokine-activated killer cytotoxicity of non-cultured melanoma cells was similar in all age groups. NK cell activity was highly correlated with levels of morphologically defined granular lymphocytes (p = 0.005) and moderately with total Leu11+ cells (p = 0.06) but not with other subpopulations of granular lymphocytes.     

Effective size of the early-run sockeye salmon Oncorhynchus nerka population of Lake Azabach'e, Kamchatka Peninsula evaluation of the effect of interaction between subpopulations within a subdivided population

The effect of subdivision on the effective size (Ne) of the early-run sockeye salmon Oncorhynchus nerka population of Lake Azabach'e (Kamchatka Peninsula) has been studied. The mode of this effect is determined by the relative productivity of the subpopulations and its magnitude, by the rate of individual migration among subpopulations and genetic differentiation. If the contributions of subpopulations (offspring numbers) are different, genetic differentiation can reduce the Ne of the subdivided population. At equal subpopulation contributions, genetic differentiation always increases the Ne of the subdivided population in comparison with a panmictic population. We have found that all sockeye salmon subpopulations of Azabach'e Lake produce equal offspring numbers contributing to the next generation. The genetic differentiation between sockeye salmon subpopulations is low, and the subdivision increases the Ne of the early-run race with reference to the sum of the effective sizes of the subpopulations by as little as 2%.     

Role of the charged amino acid residues in the cytoplasmic loop between putative transmembrane segments 6 and 7 of Na+-ATPase of an alkaliphilic bacterium, Exiguobacterium aurantiacum

When bacterial cells are subjected to stress, cellular and population characteristics can change significantly. Rapid acquisition of these properties of cells in their native physiological state is necessary in order to precisely interpret their survival modes. The multiparametric analytical capability of flow cytometry allows us to acquire these data instantaneously. In this study, the temporal cellular and population distribution analyzed by flow cytometry for periods >500 h under osmotic stress revealed that bacterial cells were diversified into more than seven different subpopulations that differed in their intracellular DNA levels. It was also revealed that individual subpopulations attained a final surviving mode devoid of any intermittent or interdependent stages within themselves. Hence, bacterial cells were subjected to rigorous spontaneous metastable (nonequilibrium) variations in their cellular and population characteristics before reaching an equilibrium state, which allowed them to prolong their survival under stress, as revealed by robust flow cytometric analysis, in their native physiological state(s).     

Functional heterogeneity among the T-derived lymphocytes of the mouse. V. Response kinetics of peripheral T cell subpopulations.

The kinetics of the proliferative response and the appearance of effectors of helper activity after stimulation by antigen were examined in T cell subpopulations. As defined in previous papers of this series, one population, T1, is short-lived after adult thymectomy (ATx), and relatively resistant to elimination by anti-thymocyte serum (ATS). Another population, T2, is long-lived after ATx, but highly sensitive to elimination by small doses of ATS. From precursors within the T2 population, effectors of specific helper activity, after priming with antigen, appeared within 1 to 2 days and reached a maximum on day 4. The responding cells reached their peak proliferative response within 24 hr after stimulation by antigen. In contrast, helper activity arising from T1 precursors first appeared on day 3 and peaked on day 5. These cells did not reach their maximal proliferative response until 60 hr after priming. These findings indicate additional useful markers for distinguishing the T1 and T2 subpopulations and are consistent with models for T cell development in which T1 cells are virgin cells and T2 cells are memory cells.     

Physical discrimination between human T-lymphocyte subpopulations by means of light scattering, revealing two populations of T8-positive cells

Light-scattering properties of human T-lymphocyte subpopulations selected by immunofluorescence were studied. Based on differences in orthogonal light scattering, two subpopulations of T8-positive cells can be distinguished. The first population (T8a) has the same orthogonal light-scattering properties as T4-positive cells, whereas the orthogonal light scattering of the second population (T8b) was about 70% larger. Orthogonal light scattering of Leu7-positive lymphocytes resembles that of the T8b population. We have studied the occurrence of the subpopulation in healthy individuals and we discuss their possible functional identification. Light-scattering properties of lymphocyte subpopulations in two patients with B-cell chronic lymphatic leukemia suggest that this observation is of clinical interest.     

Production of giant cells of Escherichia coli.

Giant cells, with volumes up to 500-fold those of normal cells, have been produced by both genetic and pharmacological means in Escherichia coli K-12. In the genetic approach, an envB or mon mutation (conferring rounded or irregular morphology) was combined with a lon mutation (block of septation after irradiation). UV irradiation and subsequent incubation for 2 to 5 h in a rich medium supplemented with 1% sodium chloride led t; production of polymorphic giant cells. In the pharmacological approach, incubation of several different strains of E. coli K-12 with the drug 6-amidinopenicillanic acid (FL1060) in the same rich medium gave rise to a homogeneous population of smoothly rounded giant cells.     

Effect of a monoclonal anti-large granular lymphocyte antibody on the human NK activity

A monoclonal hybridoma antibody of IgGIa subclass was produced by fusing NS-1 myeloma cells with spleen cells of a mouse immunized with human LGL cells. This hybridoma antibody, termed NK-8, was reactive by indirect immunofluorescence with 33% of peripheral blood LGL cells and 70% of LGL forming conjugates with K-562 cells. Monocytes, granulocytes, and other lymphocytes were nonreactive. In iodinated protein A binding assays NK-8 was nonreactive with all kinds of leukemia and lymphoma lines tested and showed activity only against LGL cells. NK-8 inhibited the LGL-mediated cytotoxicity against K-562 cells by 50 to 60% without complement and inhibited the K-562 induced interferon production from the LGL population. However, the spontaneous cytotoxicity against human skin fibroblasts was augmented if the effector cells were pretreated with NK-8.     

In vitro selection of SV40 T antigen epitope loss variants by site-specific cytotoxic T lymphocyte clones

SV40-transformed cells of C57BL/6 (B6) mouse origin (H-2b) express four distinct predominant antigenic sites, I, II, III, and IV, on SV40 large tumor (T) Ag that are recognized by SV40 T Ag-specific CTL clones. In this study, we selected SV40 T Ag-positive cell lines which had lost one or more of the antigenic sites, by in vitro cocultivation of a SV40-transformed B6 mouse kidney cell line (K-0) with SV40 T Ag site-specific CTL clones, Y-1 (site I specific), Y-2 (site II specific), Y-3 (site III specific), and Y-4 (site IV specific). All of the CTL-resistant cell lines expressed large quantities of cell surface H-2 class I Ag. K-1 cells selected by CTL clone Y-1 lost the expression of antigenic sites I, II, and III, but not site IV. K-2 and K-3 cells selected by CTL clones Y-2 and Y-3, respectively, were found to be negative for sites II and III but expressed sites I and IV. K-4 cells selected by CTL clone Y-4 lost the expression of only site IV. K-1,4 cells (sites I-, II-, III-, IV-) were selected from K-1 cells by cocultivation with CTL clone Y-4, K-2,4 cells (sites I+, II-, III-, IV-) were selected from K-2 cells by CTL clone Y-4. K-3,1 cells (sites I-, II-, III-, IV+) were selected from K-3 cells by CTL clone Y-1, and K-3,1,4 cells (sites I-, II-, III-, IV-) were selected from K-3,1 cells by CTL clone Y-4. From K-4 cells, K-4,1 cells (sites I-, II-, III-, IV-) and K-4,3 cells (sites I+, II-, III-, IV-) were selected by CTL clone Y-1 and Y-3, respectively. The antigenic site loss variant cell lines K-1, K-1,4, K-3,1 K-3,1,4, K-4,1, and K-4,3 synthesized SV40 T Ag molecules of 75, 75, 78, 78, 81, and 88 kDa, respectively. Expression of wild-type SV40 T Ag in the antigenic site loss variants by infection with SV40 or transfection with cloned SV40 DNA restored the CTL recognition sites on the variant cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Identification of early stages of T lymphocyte development in the thymus cortex and medulla

Thymocyte subpopulations with a phenotype suggesting they are early stages of T cell development in the adult mouse thymus were characterized and isolated by using multiparameter flow cytometry and sorting, in conjunction with selective killing with antibody and complement (C). The intrathymic localization of these subpopulations was assessed by dipping the thymus in fluorescent dyes to selectively label outer-cortical cells. The main phenotypic markers used were sensitivity to C-mediated lysis by the monoclonal antibody B2A2 (which spares most prothymocytes but kills most thymocytes), the expression of the T cell lineage specific markers Ly-2 and L3T4, and the levels of the common T cell antigens Ly-1 and Thy-1. A preliminary selection for cells lacking Ly-2 and L3T4, or resistant to B2A2 and C, produced a population of large cells, only 5% of all thymocytes and distinct from the typical cortical blast cells. This population of putative early thymocytes was itself heterogeneous, consisting of eight subpopulations separable by phenotype and intrathymic localization. One group of two subpopulations (B2A2-, Ly-1++, Thy-1+ and either Ly-2+ L3T4- or Ly-2- L3T4+) appeared to be of medullary location, and their phenotype suggested they could have been early members of the medullary lineages. Another group of two subpopulations (B2A2-, Ly-1++, Thy-1-, Ly-2-, L3T4- and B2A2-, Ly-1++, Thy-1+, Ly-2- L3T4-) did not show a clear localization pattern and may have represented cells in an earlier stage of transition to medullary phenotype and location. A quite different group of three subpopulations (B2A2++, Ly-1-, Thy-1-, Ly-2- L3T4-; B2A2++, Ly-1-, Thy-1+, Ly-2-, L3T4-; and B2A2++, Ly-1+, Thy-1++, Ly-2- L3T4-) was concentrated in the outer cortex and seemed to represent a series of stages of a cortical pathway, before the typical cortical blast cells. Finally, a very minor subset (0.2% of thymocytes), lacking all these markers, was concentrated in the outer cortex; this fifth group had the phenotype expected of the earliest intrathymic precursor cells. The results suggest that the separate developmental streams of cortical and medullary thymocytes may be traced back, via these minor early blast subpopulations, to common precursor cells in the outer cortex.     

The spatial and temporal expression of HNK-1 by myogenic and skeletogenic cells in the embryonic rat

The existence of phenotypic differences within a population of cells provides evidence for discrete stages in cellular differentiation and/or identifies subsets of cells with unique functional properties. The monoclonal antibody HNK-1 has been widely shown to identify subpopulations of cells in the developing nervous system. In this paper we focus on the developmental expression of HNK-1 immunoreactivity by derivatives of somitic (paraxial) mesoderm. We show that between embryonic day 12 and 14 (E12–E14) the HNK-1 epitope is transiently expressed by postmitotic myotomal cells. In E14–E17 developing vertebral columns (which are derived from somitic sclerotomal cells), HNK-1 immunolabeling was expressed by subpopulations of skeletogenic cells, including perinotochordal cells associated with the forming annulus fibrosus and cells within or adjacent to the perichondrium. Chondrocytes within forming centra and vertebral arches did not exhibit HNK-1 immunoreactivity. These results, taken together, show that the expression of the HNK-1 epitope can be used to identify subsets of myogenic and skeletogenic cells both spatially and temporally in the developing rat.     

Identification of human lymphocyte-derived lymphotoxins with binding and cell-lytic activity on NK-sensitive cell lines in vitro

Supernatants obtained from lectin-restimulated, preactivated, human peripheral blood lymphocytes rapidly released (5–24 hr) high levels of lymphotoxin (LT) activity in vitro. Peripheral blood lymphocytes were preactivated by coculturing with either fetal calf serum or with allogeneic continuous B-cell lines (LCCL) which were treated with mitomycin C. These Supernatants contained a population of L-929 cell-lytic LT forms which also selectively bind to the NK-sensitive K-562 cell. However, lytic LT forms for L-929 cells from cPBL and LCCL cultures did not bind to the NK-sensitive MOLT-4 or NK-resistant Raji cells. Additional studies reveal these supernatants contain a second set of LT forms which have cell-binding and cell-lytic activity detectable on MOLT-4 and K-562 cells in a 12 to 18 hr ⁵¹Cr-release assay. Cell-lytic form(s) for the MOLT-4 and K-562 cells were not stable for more than a week at ?20°C. These findings indicate that materials with LT activity are heterogeneous with respect to their capacity to recognize common and discrete cell-surface components on different types of target cells in vitro.     

Progenitor cells in the thymus: most thymus-homing progenitor cells in the adult mouse thymus bear Pgp-1 glycoprotein but not interleukin-2 receptor on their cell surface

Pgp-1-positive and interleukin-2-receptor (IL-2R)-positive cells are both minor (less than 5%) subpopulations within the adult thymus. A thymocyte population enriched for these two cell types, obtained by killing the bulk of thymocytes with anti-Thy-1 antibody and complement, contains thymus-homing progenitor cells which can transiently repopulate the thymus of irradiated recipients. Using two-color immunofluorescence, we demonstrate that the Pgp-1+ and IL-2R+ cells present in this enriched population represent largely nonoverlapping subsets, although some cells do express both markers. We also show by depletion of these two cell types that the bulk of the thymus-homing progenitors present in this enriched population are found in the Pgp-1+ population, and not in the IL-2R+ population. We discuss the relationship between the thymus-homing progenitors in this depleted thymus subpopulation and the thymus-homing progenitors present in the thymus as a whole.     

Size-dependent B lymphocyte subpopulations: relationship of cell volume to surface phenotype, cell cycle, proliferative response, and requirements for antibody production to TNP-Ficoll and TNP-BA

Murine splenic B lymphocytes were separated into size-dependent subpopulations by using counterflow centrifugation. Spleen cells were rigorously depleted of T lymphocytes to yield a population of cells that were greater than 90% surface immunoglobulin (Ig)-positive and that had a mean cell volume of 136.6 +/- 3.3 microns. From this population, five fractions of cells were obtained with mean cell volumes that ranged from 115.8 +/- 3.7 microns in fraction 1 to 168.0 +/- 6 microns in fraction 5. The cells in these five subpopulations were characterized by analysis on a fluorescence-activated cell sorter after staining with acridine orange to evaluate RNA and DNA content, and with fluorescein-conjugated anti-mu, anti-delta, and anti-Ia antibodies to evaluate their surface membrane phenotypes. DNA analysis revealed that virtually all of the cells in fractions 1 to 4 had 2 N DNA. Between 7 and 21% of fraction 5 cells were either in S-phase or contained 4 N DNA. In contrast, RNA content increased through the fractions, suggesting a transition from G0 to G1 in the subpopulations with increasing B cell size. As another measure of cell activation seen with increasing cell size, we observed a progressive increase in the expression of surface Ia and a decrease in the expression of surface IgD. In the absence of in vitro stimulation, the larger cells showed significantly higher levels of thymidine incorporation. When polyclonal B cell activators such as LPS or anti-Ig antibody were added, peak proliferative responses were similar in all of the fractions, but the time necessary to achieve a maximal response was shorter for the larger-sized cell subpopulations than it was for the smaller-sized cell subpopulations. Unprimed, size-dependent B lymphocyte subpopulations exhibited spontaneous or "background" antibody formation that occurred primarily in the subpopulations containing the largest cells. T cell factors present in EL4 supernatant enhanced the efficiency of in vitro differentiation of these same subpopulations. When cultured in the absence of T cell help, the thymus-independent type 1 (TI-1) antigen TNP-Brucella abortus (TNP-BA) or the thymus-independent type 2 (TI-2) antigen TNP-Ficoll induced the largest anti-TNP plaque-forming cell (PFC) responses in the fractions containing intermediate-sized cells, suggesting that in vitro, antigen-specific responses came primarily from B cells that have been influenced in vivo to leave their small resting state. The subpopulations containing the smallest size B cells required the presence of both a TI antigen and EL4 supernatant for efficient differentiation.(ABSTRACT TRUNCATED AT 400 WORDS)     

Separation of isolated hamster intestinal epithelial cells by velocity sedimentation on Ficoll gradients

Isolated hamster intestinal epithelial cells can be separated by velocity sedimentationion on 2–10% Ficoll gradients into three subpopulations of cells which differ in morphology, biochemistry, physiology, and membrane components. These subpopulations are not pure but are enriched in a single cell type to the extent that differences in cell function can be observed. The proliferative crypt cells are separated from the digestive-absorptive villus cells. A third subpopulation with a distinctive morphology is also obtained. Quantitation of DNA recoveries from the gradients indicates that this population constitutes approximately one-third of the epithelial cell population. These carrot-shaped cells are found adjacent to the digestive-absorptive columnar epithelial cells on the villus. The two types of villus cells differ in glycolipid or glycoprotein components of the brush border as shown by lectin binding experiments with the isolated cells. The gradient data also suggest that only one-third of the intestinal epithelial cell population is responsible for most monosaccharide absorption in hamster small intestine.