Cell Surface Antigen Phenotype Research Articles

Comparison of biological properties of umbilical cord-derived mesenchymal stem cells from early and late passages: Immunomodulatory ability is enhanced in aged cells

Mesenchymal stem cells (MSCs) are a potential source of adult stem cells for cell-based therapeutics due to their substantial multilineage differentiation capacity and secretory functions. No information is presently available regarding the maintenance of immunosuppressive properties of this cell type with repeated passages. It was therefore the aim of the present study to analyze the biological properties, particularly the immunoregulatory effect, of MSCs from late passages. The differences between young and old MSCs in morphology, cell surface antigen phenotype, proliferation, gene expression and immunomodulatory ability were investigated. The results of the current study demonstrated that with the passage of cells, senescent MSCs displayed a characteristically enlarged and flattened morphology, different gene expression profiles and stronger immunosuppressive activities. Increased interleukin-6 production may be a possible underlying mechanism for this enhanced immunomodulatory ability of MSCs. These findings suggest that aged MSCs may provide a treatment option for patients with graft versus host disease and other diseases associated with dysregulation of the immune system.

The effects of adenoviral transfection of the keratinocyte growth factor gene on epidermal stem cells: an in vitro study.

Epidermal stem cells (ESCs) are characterized as slowcycling, multi-potent, and self-renewing cells that not only maintain somatic homeostasis but also participate in tissue regeneration and repair. To examine the feasibility of adenoviral vector-mediated keratinocyte growth factor (KGF) gene transfer into in vitro-expanded ESCs, ESCs were isolated from samples of human skin, cultured in vitro, and then transfected with recombinant adenovirus (Ad) carrying the human KGF gene (AdKGF) or green fluorescent protein gene (AdGFP). The effects of KGF gene transfer on cell proliferation, cell cycle arrest, cell surface antigen phenotype, and β-catenin expression were investigated. Compared to ESCs transfected with AdGFP, AdKGFtransfected ESCs grew well, maintained a high proliferative capacity in keratinocyte serum-free medium, and expressed high levels of β-catenin. AdKGF infection increased the number of ESCs in the G0/G1 phase and promoted ESCs entry into the G2/M phase, but had no effect on cell surface antigen phenotype (CD49f(+)/CD71(-)). The results suggest that KGF gene transfer can stimulate ESCs to grow and undergo cell division, which can be applied to enhance cutaneous wound healing.

Effects of low frequency electromagnetic field on proliferation of human epidermal stem cells: An in vitro study

To investigate the effects of low frequency electromagnetic fields (EMF) on the proliferation of epidermal stem cells, human epidermal stem cells (hESC) were isolated, expanded ex vivo, and then exposed to a low frequency EMF. The test and control cells were placed under the same environment. The test cells were exposed for 30 min/day to a 5 mT low frequency EMF at 1, 10, and 50 Hz for 3, 5, or 7 days. The effects of low frequency EMF on cell proliferation, cell cycle, and cell-surface antigen phenotype were investigated. Low frequency EMF significantly enhanced the proliferation of hESC in the culture medium in a frequency-dependent manner, with the highest cell proliferation rate at 50 Hz (P < 0.05). Exposure to a low frequency EMF significantly increased the percentage of cells at the S phase of the cell cycle, coupled with a decrease in the percentage of cells in the G1 phase (P < 0.05) but the effect was not frequency dependent. The percentage of CD29(+) /CD71(-) cells remained unchanged in the low frequency EMF-exposed hESC. The results suggested that low frequency EMF influenced hESC proliferation in vitro, and this effect was related to the increased proportion of cells at the S phase.

Effects of 50 Hz pulsed electromagnetic fields on the growth and cell cycle arrest of mesenchymal stem cells: an in vitro study

Mesenchymal stem cells (MSCs) are capable of self-renew and multipotent differatiation which allows them to be sensitive to microenvironment is altered. Pulsed electromagnetic fields (PEMF) can affect cellular physiology of some types of cells. This study was undertaken to investigate the effects of PEMF on the growth and cell cycle arrest of MSCs expanded in vitro. To achieve this, cultured of normal rat MSCs, the treatment groups were respectively irradiated by 50 Hz PEMF at 10 mT of flux densities for 3 or 6 h. The effects of PEMF on cell proliferation, cell cycle arrest, and cell surface antigen phenotype were investigated. Our results showed that exposed MSCs had a significant proliferative capacity (P < 0.05) but the effect of PEMF for 3 and 6 h on cell growth was not different (P>0.05) at an earlier phase after PEMF treatment. Exposure to PEMF had a significant increase the percentage of MSCs in G1 phase compare with the control group, with a higher percentage of cells in G1 phase exposed for 6 h then that for 3 h. At the 16th hour after treatment, PEMF had no significant effect on cell proliferation and cell cycle (P>0.05). These results suggested that PEMF enhanced MSCs proliferation with time-independent and increased the percentage of cells at the G1 phase of the cell cycle in a time-dependent manner, and the effect of PEMF on the cell proliferation and cell cycle arrest of MSCs was temporal after PEMF treatment.

Leukemia stem cells in 2010: Current understanding and future directions

Myeloid leukemias are clonal disorders originating in a primitive multipotential hematopoietic cell and characterized by aberrant proliferation, differentiation and maturation of leukemic progenitors and precursor cells. These diseases are the result of multiple genetic and epigenetic events, although the nature and number of events vary widely among patients. For over four decades, studies have identified sub-populations of leukemic cells possessing different functional capabilities. Investigators have struggled to understand the origin and significance of this heterogeneity. The stem cell model for myeloid malignancies has offered one potential explanation. Since 1994, experimental data supporting the presence of leukemia stem cells has been reported and validated in numerous studies. We will review the history of the model, data from the past decade supporting the stem cell model for myeloid malignancies, more recent data regarding patient specific variability in leukemic stem cell surface antigen phenotype and the impact the stem cell model has on the care of patients with myeloid malignancies.

Use of cell surface antigen phenotype in guiding therapeutic decisions in chronic myelomonocytic leukemia

The myelodysplastic syndromes are a heterogeneous group of hematopoietic stem cell diseases in which both diagnosis and prognosis are determined by cellular morphologic criteria. In some patients, prognosis is poor due to proliferation of immature cells, i.e. development of acute leukemia. An important clinical decision for patients with myelodysplastic syndromes is whether to treat with supportive care or to use cytoreductive drugs to control the proliferative component of these illnesses. Two cases of chronic myelomonocytic leukemia are presented where cell surface antigen phenotype analysis showed characteristics suggestive of proliferative disease and the patients were treated and obtained remission with cytoablative therapy. Cell surface marker analysis may be useful in guiding therapeutic decisions in myelodysplasia.

A study of granulated metrial gland cell differentiation in pregnant, macrophage-deficient, osteopetrotic (op/op) mice.

A population of uterine natural killer (NK) cells, commonly called granulated metrial gland (GMG) cells, differentiates in the mouse uterus during normal pregnancy. Little is known regarding the process of differentiation of GMG cells or of other NK cell subsets. It has been suggested that macrophage precursors, under the combined influences of the cytokine growth factors colony stimulating factor-1 (CSF-1) and interleukin-2, become NK-cell like in morphology, pattern of target cell lysis and surface antigen phenotype. Mice expressing the mutation osteopetrosis (op/op) are unable to produce the cytokine CSF-1. To determine whether CSF-1 is required for the successful differentiation of uterine NK cells, implantation sites in pregnant, op/op mice were studied histologically. GMG cell differentiation appeared to progress normally in op/op mice studied between days 7 and 14 of gestation. Thus, the growth factor CSF-1 is not required for differentiation of the uterine NK cell subset known as GMG cells and probably GMG cells do not differentiate from macrophage precursor cells which are deficient in op/op mice.

Characterization of a subset of human B lymphocytes interacting with natural killer cells.

Tonsil B cells were analyzed for their capacity to interact directly with NK cells in vitro. A specific, direct interaction between NK cells and B cells could be detected by direct conjugation and by cold target inhibition using the B lymphoblastoid cell line BJA.B as a labeled target. The data further suggest that the B cell interaction with NK cells specifically activates the NK effectors and induces their production of IFN-gamma. The NK-interactive population of tonsil B cells were characterized as low-buoyant density cells (by Percoll gradient fractionation) that stained more brightly with Hoechst 33342, both characteristics of activated B cells. Immunofluorescent staining of NK cell-B cell conjugates allowed determination of the cell-surface antigenic phenotype of conjugate-forming B cells. B cell targets were ICAM-1bri, 4F2+, TfR+, CD32+, BB1+, and CD77-. They tended to be CD38-, but overlapped the CD38+ population. No correlation was seen with CD37, CD44, CD75, CD76, HC2, or Ig kappa. This phenotype is most consistent with a late activation stage of differentiation, just before and overlapping the expression of CD38. These B cells do not appear significantly sensitive to NK-mediated cytolysis, suggesting that NK cell cytokine synthesis and secretion (e.g., IFN-gamma) may be more important in the NK cell regulation of the humoral response.

The surface antigen phenotype of human embryonal carcinoma cells: modulation upon differentiation and viral infection.

The cell surface provides a rich source of developmentally controlled molecules that are of interest to several branches of basic cell biology and clinical medicine. Originating from studies of ontogeny and function in the immune system, the notion of cell surface antigen phenotype, defined by the pattern of expression of several “differentiation antigens” (Bennett et al. 1972; Boyse and Old 1978), also provides a valuable tool for following the progression of cell types in other complex differentiating systems such as those presented by teratocarcinomas. We have used the expression of a variety of surface antigens to provide objective criteria for the identification of human embryonal carcinoma (EC) cells, the stem cells of teratocarcinomas; we have also used changes in the expression of such antigens to study the differentiation of a pluripotent human EC cell line, TERA 2 (Andrews et al. 1984b). Extension of these observations to a clinical setting could provide the basis for improved histological classification of human germ cell tumors. Moreover, as we report here, the shedding of certain surface antigens characteristic of EC cells and their detection in the serum of patients could provide a useful additional means of monitoring the progress of therapy.

Rat monoclonal antibodies produced against rat colorectal adenocarcinomas define tumor‐ and colon‐associated, auto‐immunogenic antigens

Monoclonal antibodies (MAbs) were derived from rats immunized against allo- and syngeneic rat colon carcinomas. Screening was performed by immunohistochemistry modified for MAbs of the same species as the tissue used for frozen sections. From 2 fusions, several MAbs were found that bound to syngeneic tumor tissue but showed little or no staining of normal adult tissues. Another group of MAbs demonstrated an intracellular staining of tumor cells and staining of mucus and goblet cells in the distal 2/3 of the normal colon. A third group of MAbs showed staining of both tumor and normal colon tissue. Staining patterns were reproduced with 6 MAbs selected from the first 2 groups after purification and biotinylation. One MAb, 10B12, recognized an antigen expressed in 10/11 colon carcinomas, the lowest parts of colonic crypts, intracellularly in a subpopulation of pancreatic acinar cells and mucus in antrum. It was used for affinity purification of a high-molecular-weight antigen, to which 3 of the other rat MAbs also bound. This antigen was also bound by antibodies to blood group A and isogloboseries carbohydrate determinants. Competition with the labelled 10B12 MAb for binding to the purified antigen was demonstrated in sera of tumor-bearing and immune rats. Thus, this high-molecular-weight glycoprotein expressed both auto-antigenic, tumor-associated and tissue-type-restricted epitopes. The 10B12 MAb homogeneously stained the majority of colorectal carcinomas and the antigenic determinant was expressed on the cell surface of 12/13 tissue-cultured colon-carcinoma clones. Modulation of the cell-surface antigen phenotype by recombinant rat interferon-gamma markedly increased expression of this determinant.

Diethyldithiocarbamate, a novel immunomodulator, prolongs survival in autoimmune [formula omitted] mice

MRL- lpr lpr mice die at an early age from a spontaneously developing systemic lupus erythematosus-like disease and are characterized by massive lymphadenopathy, hyperproliferation of Lyt-2 L3T4 (null) T cells, decreased responses to mitogens, thymic atrophy, and very high serum autoantibody levels. Diethyldithiocarbamate (DTC), an immunomodulator suggested to enhance T cells, was used to treat 12-week-old female MRL- lpr lpr mice (25 mg/kg/week). DTC treatment significantly prolonged survival (50% mortality, 43 weeks vs 20 weeks). Increased survival was associated with decreased lymphadenopathy, decreased proliferation of null cells, restoration of impaired mitogen responses, decreased thymic atrophy, and decreased serum levels of anti-DNA and anti-histone antibodies. Studies of cell surface antigen phenotype demonstrated increased expression of Lyt-2 and macrophage surface antigens. No effect on L3T4 single staining cells was observed. These results show that DTC significantly alters the disease course in these mice and suggest that DTC may be a useful treatment for autoimmune disease.

Expression of DNA Polymerase α and Leu3a Molecules in Growing and Saturated Cultures of Human Leukemic Cells: Phenotype Analysis of Proliferative Cells by Flow Cytometry

A flow cytometric method to analyze phenotypes of proliferative cells was developed using human leukemic cell line MOLT 4. A nuclear protein, DNA polymerase α (pol α), was selected as a marker for proliferative cells, and Leu3a molecule as a cell‐surface antigen phenotype marker of the cells. The procedure involved the simultaneous use of fluorescein‐conjugated anti‐pol α antibody, developed by us, and commercially available phycoerythrin‐conjugated anti‐Leu3a antibody. The optimal fixative for both proteins was phosphate‐buffered 2% paraformaldehyde. The pol α‐positive population in logarythmically growing MOLT 4 cells was estimated, by flow cytometry, to be ca. 95%. A sharp flow cytometry histogram with a strong pol α‐linked fluorescence was observed. On the other hand, the pol α‐positive population in the saturated culture was ca. 70%, with weaker pol α‐linked fluorescence. Thus, the population of pol α‐positive cells and the amount of pol α in cells was dependent on the cell density of the culture. In contrast, ca. 90% Leu3a‐positive populations with similar flow cytometry histograms were seen in either growing or saturated states, suggesting that expression of Leu3a was independent of cell density. The flow cytometric method using fluorescein isothiocyanate‐conjugated anti‐pol α antibody is useful for detecting proliferative fractions of free tumor cells, such as leukemic cells. Furthermore, analysis of the phenotype of the proliferative or non‐proliferative cells became easier by simultaneous labeling with antibodies against pol α and phenotype‐speciflc proteins.

Immunophenotypic profile of CNS lymphoma: a review of eighteen cases.

The cell surface antigenic phenotype of 18 cases of central nervous system (CNS) large-cell lymphoma (14 primary, four secondary) was examined by an immunoperoxidase technique using antibodies that identify B cell restricted and associated antigens. All cases were shown to be of B cell origin by virtue of the expression of monotypic immunoglobulin (Ig) (16 IgM, two IgG) and the pan B cell antigen B1 (CD20). A panel of monoclonal antibodies directed against B cell restricted and associated activation antigens including B5, Blast-1, Blast-2 (CD23), BB1, interleukin 2 receptor (IL2R, CD25), T9 (transferrin receptor) and TNK-TAR (4F2) was used on 12 of the cases. The majority expressed T9 and TNK-TAR. Blast-1 was expressed by less than half the cases and Blast-2 and B5 by one of 12 cases each. This is in contrast to 10 non-CNS diffuse large cell lymphomas where B5 and Blast-1 were present on all cases. This study confirms previous observations that primary CNS large cell lymphomas are of B cell derivation. Moreover, the differences in expression of B cell activation antigens on CNS large cell lymphomas as compared to non-CNS lymphomas raise the possibility that a subset of neoplastic B cells may have unique tropism for the CNS.

Establishment of a murine leukemia L1210-specific T-cell clone displaying novel in vivo anti-tumor activity.

The murine leukemia L1210-specific cytotoxic T-cell clone K7 was established by repeated antigenic stimulation of spleen cells of L1210-immune CD2F1 mice in long-term culture. K7 possessed L1210-specific cytolytic activity detectable by the 51Cr-release test, but lost its cytolytic activity about 100 days after initiation of culture (designated as clone K7L). Clone K7L retained its L1210-specific tumor-growth-inhibitory activity independently of culture supplementation with IL-2. K7L possessed the cell-surface antigenic phenotype of cytotoxic T cells, Thy-1+, Lyt-1-, Lyt-2+. This clone K7L displayed surprisingly strong activity in specifically inhibiting in vivo tumor growth of L1210 by day 5 in the peritoneal cavity of CD2F1 mice when injected together with 10(5) tumor cells (more than 90% inhibition at E/T = 5), and prolonged survival times of most of the mice for more than 60 days, whereas control mice inoculated with L1210 alone died on day 9-17. This unique in vivo anti-tumor activity was not correlated to the in vitro cytolytic activity. Nevertheless, bystander inhibitory effect on the growth of third-party tumor cells (P388) was not seen in mice injected with a mixture of L1210 and P388 together with K7L, suggesting that K7L inhibited L1210 growth by direct cell-to-cell interaction. Host cells such as X-irradiation (800R)-sensitive lymphocytes and Carrageenan-sensitive macrophages were not involved in the early growth inhibition of L1210 by K7L.

Fractionation of human bone-marrow mononuclear cells with peanut agglutinin: Phenotypic characterization with monoclonal antibodies

Bone marrow mononuclear cells were fractionated by affinity chromatography on immobilized peanut agglutinin (PNA). The resulting PNA + fraction represented 10% of the total cell number. Twenty percent of the cells within the PNA + compartment compartment coexpressed the T6, Ia, Mo1, and My4 differentiation antigens and possesed Fc and C3 receptors. The similarity in cell surface antigen phenotype led us to hypothesize that this subset may be a cellular precursor of dentritic cells found in the skin (Langerhans cells) or in the parenchimal organs of the body (D-cells)

Generation, propagation, and variation in cloned, antigen-specific, Ia-restricted cytolytic T-cell lines.

Since the demonstration that B lymphocytes bear surface immunoglobulin (Ig), while helper T lymphocytes are surface Ig negative but bear the T-cell differentiation antigen Thy-1, attempts have been made to correlate the function of a T cell with the expression of differentiation antigens by that cell. In the mouse and in man, it has been found that the majority of helper T cells bear the T4 (or L3T4a) marker, while suppressor and cytolytic T cells bear the T8 or Lyt-2 surface molecule (Cantor and Boyse 1976; Jandinski et al. 1976; Rheinherz et al. 1979). However, many studies, especially those employing cloned T-cell populations, appear to violate these “rules.” Thus, cells with helper, suppressor, or cytolytic capabilities have been assigned to either of these populations by several investigators (Swain et al. 1981; Thomas et al. 1981). In this report, we will dis-cuss our current work on cytolytic T cells that are antigen-specific, la-restricted, and L3T4a positive (Tite and Janeway 1984a, b; Tite et al. 1985). Such cells also manifest at least some of the functions associated with helper T cells. Our studies indicate that the critical determinant of the function of a T cell is the antigen- nonspecific molecules it produces upon activation by antigen:MHC, not its cell surface antigen phenotype or its specificity for a particular self-MHC molecule. We will present evidence that demonstrates that such cells are found amongst normal T cells, and that previous attempts to demonstrate such cells probably failed for technical reasons having to do with assay conditions and target-cell susceptibility.KeywordsSpleen Cell51Cr Release AssayCytolytic FunctionDrain Lymph Node CellMitogenic LectinThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Differentiation of Human T Lymphocytes: II. Phenotypic Difference in Skin and Blood Malignant T-Cells in Cutaneous T-Cell Lymphoma

The cell surface antigen phenotype of circulating and skin malignant T-cells in patients with cutaneous T-cell lymphoma were studied. The mature T-cell antigen phenotype of the malignant T-cells was identical for circulating and skin malignant T-cells. In contrast, skin malignant T-cells expressed the immature human T-cell antigen Thy-1, surface membrane transferrin receptors, and Ia-like determinants while circulating malignant T-cells did not express these antigens. Skin infiltrating T-cells in benign dermatoses also expressed Thy-1 and Ia-like determinants. These observations support the notion that the skin is a major site of extrathymic T-cell differentiation and may promote phenotypic changes in circulating T-cells that home to skin.

The binding of Maclura pomifera lectin to cells of the T-lymphocyte lineage in the rat

The tissue, anatomic, and developmental distribution of Maclura pomifera (MP) lectin binding to rat lymphoid cells was examined. Analysis was performed by immunofluorescence microscopy and by the fluorescence-activated cell sorter. Comparison with anti-rat Ig to detect B cells and the monoclonal antibodies W3/13, W3/25, and OX 8 to detect T cells revealed that the MP lectin reacted with all T cells and not B cells in spleen and lymph node of young adult rats. The lectin also bound selectively to the thymus-dependent areas in frozen sections of spleen and lymph node. Using the MP lectin in conjunction with anti-Thy1 antibody and the monoclonal antibodies, W3/25 and OX 8, four T-cell subpopulations in spleen and lymphnode were identified on the basis of their cell surface antigenic phenotype. The T-cell developmental distribution of MP binding revealed that 100% of normal and neoplastic thymocytes bound the lectin whereas approximately 25% of TdT + bone marrow cells, putative thymocyte progenitors, were MP +. Thus, the MP lectin is a nonimmunoglobulin reagent which binds to prethymic, thymic, and post-thymic cells of the T-lymphocyte lineage. Affinity chromatography experiments indicated that the MP lectin binds, at least in part, to the major thymocyte cell surface glycoprotein which is recognized by the W3/13 monoclonal antibody.

Differentiation-Linked Expression of Cell Surface Markers on HL-60 Leukemic Cells

The cell surface antigenic phenotype of HL-60, a human acute promyelocytic leukemia cell line, has been analyzed before and after maturation induction with dimethylsulfoxide (DMSO) using a panel of markers including a "library" of monoclonal antibodies and "conventional" antisera in conjunction with the fluorescene-activated cell sorter. HL-60 cells express granulocyte and "leukocyte" differentiation antigens but not antigens of the lymphoid, platelet, and erythroid lineages. DMSO-induced morphological maturation was found to be associated with a decrease in the proportion of cells in mitotic cycle, induction of C3d receptors, increased expression of granulocytic and leukocyte antigens, and diminished expression of HLA-A,B,C and beta 2-microglobulin determinants. HL-60 cells have no detectable expression of HLA-DR-associated determinants as assayed by rabbit anti-p28,33 monoclonal anti-HLA-DR (monomorphic determinant), and HLA-DRw typing alloantisera. The relationship of these changes in cell surface properties to normal granulocytic differentiation is discussed.

Differentiation-linked expression of cell surface markers on HL-60 leukemic cells

The cell surface antigenic phenotype of HL-60, a human acute promyelocytic leukemia cell line, has been analyzed before and after maturation induction with dimethylsulfoxide (DMSO) using a panel of markers including a “library” of monoclonal antibodies and “conventional” antisera in conjunction with the fluorescene-activated cell sorter. HL-60 cells express granulocyte and “leukocyte” differentiation antigens but not antigens of the lymphoid, platelet, and erythroid lineages. DMSO-induced morphological maturation was found to be associated with a decrease in the proportion of cells in mitotic cycle, induction of C3d receptors, increased expression of granulocytic and leukocyte antigens, and diminished expression of HLA-A,B,C and beta 2-microglobulin determinants. HL-60 cells have no detectable expression of HLA-DR-associated determinants as assayed by rabbit anti-p28,33 monoclonal anti-HLA-DR (monomorphic determinant), and HLA-DRw typing alloantisera. The relationship of these changes in cell surface properties to normal granulocytic differentiation is discussed.