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Biomedical subjects

J W Mier

Publications and source records attributed to J W Mier.

85 records · Page 5Linked to original sources
PubMed1982

Long-term human cytolytic T-cell lines allospecific for HLA-DR6 antigen are OKT4+.

Human allospecific cytotoxic T lymphocyte lines were established by weekly stimulation of peripheral blood lymphocytes with allogeneic Epstein-Barr virus-transformed lymphoblastoid cell lines in the presence of interleukin 2. The cytotoxic T lymphocyte lines were stimulated by either JY (which expresses HLA-A,B and -DR) or Daudi (which expresses HLA-DR but not -A,B antigens). Specificity of the effector cell lines was determined by antibody blocking and by patterns of cytolysis against a panel of target cell lines. The phenotype of the effector cells was identified by both negative (antibody and complement lysis) and positive (fluorescence-activated cell sorter) selection. The anti-JY lines only recognized targets bearing HLA-A2, B7 antigens and were composed exclusively of OKT4-8+ effector cells. The anti-Daudi lines, on the other hand, specifically recognized targets bearing DR6 and eventually contained only OKT4+8- cells. Thus, stimulation by Daudi cells can result in the generation of OKT4+ CTL lines that are allospecific for DR6 antigen.

Antigens, Surface↗
PubMed1982

The purification and properties of human T cell growth factor.

Human T cell growth factor (TCGF) has been purified more than 800-fold from serum-free lymphocyte conditioned media by utilizing ion exchange chromatography with DEAE-Sepharose, gel filtration with Ultrogel AcA54, and preparative SDS-polyacrylamide gel electrophoresis. This mitogenic protein, which is released by T cells into the incubating media after exposure to a lectin (PHA), has a m.w. of 13,000 as determined by SDS-PAGE and 20,000 to 25,000 on gel filtration and has an isoelectric point of 6.8. The material extracted from acrylamide gels is a single band (or two nearly superimposed bands) when rerun on analytical SDS gels. Partially purified material is unstable even at -70 degrees C and requires the addition of bovine serum albumin or polyethylene glycol to maintain biologic activity. It is sensitive to proteolytic digestion but resistant to nucleases and thiol-reducing agents such as dithiothreitol. Human TCGF can be reversibly denatured with urea or SDS. Material extracted from acrylamide gels has been shown to sustain T lymphoblasts in tissue culture. In contrast to lectins, certain antigens, and crude lymphocyte conditioned media, purified TCGF does not initiate lymphocyte blastogenesis but is a highly selective mitogen for T cells previously activated by exposure to lectins or antigens.

Chemical Phenomena↗
PubMed1981

Human cutaneous T cell lymphoma and leukemia cell lines produce and respond to T cell growth factor.

Three cell lines of mature T cell origin derived from patients with cutaneous T cell lymphoma-leukemias (CTCL) were found to be constitutive producers of T cell growth factor (L-TCGF). These are the first reported human cell lines which constitutively produce TCGF. Biologically active TCGF could also be eluted from the surface of these cells using an acid glycine buffer under conditions that maintained cell viability, and subcellular fractionation showed that almost all the TCGF activity was associated with the plasma membrane. Over 30 other human hematopoietic cell lines derived from other disorders were unable to produce TCGF even after induction, and their acid eluates did not contain TCGF activity. L-TCGF from CTCL lines had the same biological activity as TCGF obtained from normal leukocytes (N-TCGF) in that they both supported the long-term growth of normal T cells only after the cells were previously activated by antigen or lectin. Both L-TCGF and N-TCGF increased the rate of proliferation of TCGF-independent and TCGF-dependent CTCL cell lines. The same three factor-independent cell lines that released TCGF adsorbed TCGF in a cell-concentration, time-, and temperature-dependent manner. Since the CTCL cell lines produce TCGF, adsorb TCGF, and increase their proliferative rate in response to TCGF or a related molecule, it is suggested that this endogenously produced factor plays a role in maintaining the abnormal proliferation of these cells in culture as permanently growing cell lines independent of exogenous TCGF. However, this does not mean that this is an essential aspect of neoplastic transformation. Since it is unusual to develop these cell lines in the absence of the continuous need for added TCGF, "autostimulation" may be one of the many unusual variant phenotypic properties sometimes associated with neoplastic cells that gives them a selective advantage for in vitro growth.

Adsorption↗
PubMed1980

Human T-cell growth factor: parameters for production.

Using conditioned media (CM) from phytohemagglutinin (PHA)-stimulated peripheral blood lymphocytes (PBL) we observed long-term selective growth of T-cells from normal human donors. This T-cell growth was continuously dependent on addition of a factor called T-cell growth factor (TCGF). The optimal method for preparing highly active CM from single donor PBL involves the addition of mitomycin C-treated B-lymphoblastoid cell lines to the mixture of PBL and PHA. A number of different cell lines greatly augmented the production of TCGF in 18/18 cases. Preparation of plasma membranes from the Daudi cell line could replace the intact cells in the production of TCGF but those from the cell line, Molt-4, could not. Since the cell surface of Daudi possesses HLA-D antigens but not HLA-A, B, and C, and Molt-4 has HLA-A and B and not HLA-D, it is possible that the Ia antigens (HLA-DRw in man) are important in the release of TCGF. Using this method for growth factor production, an analysis was made concerning the events necessary for lymphocyte activation and the requirements for production and release of TCGF. Removal of PHA 12 h after incubation had no effect on lymphocyte transformation but decreased TCGF release by 90%. In addition, colchicine and cytosine arabinoside inhibited DNA synthesis but had no effect on TCGF release. Little or no TCGF activity was present after cellular protein synthesis was inhibited by puromycin and cycloheximide. These results suggest that TCGF production: a) requires protein synthesis; b) requires binding of the stimulating agent; c) can occur in a non-dividing cell, probably a terminally differentiated T-cell, without the need for cellular proliferation; and d) needs the assistance of an adherent cell which probably is a monocyte-macrophage. The ability to produce TCGF from single human donors will allow better understanding of the nature and action of TCGF.

Cell Division↗
PubMed1980

Purification and some characteristics of human T-cell growth factor from phytohemagglutinin-stimulated lymphocyte-conditioned media.

Human T-cell growth factor (TCGF), a mitogenic protein that appears in the media of cultured lymphocytes after phytohemagglutinin-stimulation, has been purified more than 400-fold from serum-free conditioned media by using a sequence of ion exchange chromatography and gel filtration. The purified growth factor elutes as a broad peak from DEAE-Sepharose, focuses diffusely at a pH of about 6.8 on isoelectric focusing (suggesting heterogeneity in electrical charge), has an estimated molecular weight of approximately 23,000 as judged by gel filtration (12,000-13,000 on Na-DodSO4/polyacrylamide gel electrophoresis), is resistant to DNase and RNase, is degraded by trypsin, and does not adhere to any of several lectin-Sepharoses. These characteristics indicate that it is nonglycosylated and protein in nature. The activity of the factor determined by cell counts or [3H]thymidine incorporation in human T lymphoblasts, is stable at room temperature in crude conditioned media, but the partially purified factor requires the addition of albumin or polyethylene glycol to maintain stability. Unlike the crude conditioned media, the purified factor lacks colony-stimulating activity and, unlike lectins, antigens, and crude conditioned media, it does not initiate blastogenesis in peripheral blood lymphocytes but is a selective mitogen for T cells that have undergone blast transformation secondary to exposure to a lectin or antigen. This indicates that the factor is a second signal in the T-cell immune response. The partially purified factor has been used to selectively grow several human T-cell lines, including cells that are cytotoxic to a variety of target cells.

Culture Media↗
PubMed1980

T-cell lines established from human T-lymphocytic neoplasias by direct response to T-cell growth factor.

Long-term growth of lymphoblastoid T cells from tissue samples from six of six patients with cutaneous T-cell lymphoma (CTCL) and six of six patients with acute T-lymphoblastic leukemia (ALL) has been achieved by using partially purified mitogen-free human T-cell growth factor (pp-TCGF). One cell line, CTCL-2, is now independent of added growth factor; the others continue to show absolute dependency on its presence. All lines have been in continuous culture for at least 4 months and some for > 1 year. They are erythrocyte-rosette positive and are negative for Epstein-Barr virus nuclear antigen. Most of the lines are negative for Fc and complement receptors and for surface immunoglobulin except that CTCL-1 and CTCL-2 have some cells positive for these cell surface markers. Results of histochemical studies on these cell lines are similar to the known patterns for fresh cells from their disease of origin. Cell line CTCL-3 has an abnormal karyotype, but no detectable chromosomal abnormalities were found in the other lines, consistent with the karyologic features of their clinical sources. Because T cells from normal donors do not respond to pp-TCGF unless the cells are first "activated" by a lectin mitogen such as phytohemagglutinin or an antigen, the direct response to pp-TCGF of T cells from patients with T-cell neoplasias suggests that the cell lines represent a transformed neoplastic cell population. Although some of the cell lines may be normal T cells activated by the malignant cells, the morphologic and histochemical properties of the cell lines, the abnormal karyotype of CTCL-3, and the independent growth of CTCL-2 support the conclusion that most of these cell lines are of malignant origin.

Antigens, Surface↗
PubMed1980

Development and characterization of allospecific long-term human cytolytic T-cell lines.

Two long-term human cytolytic T lymphocyte (CTL) lines (VE and JR), whose cytolytic activity was dependent upon both irradiated JY cells (the stimulating alloantigen) and T-cell growth factor, were established. These lines were monitored in culture for 6-8 months. Both lines were specific for HLA-A or B antigens or both and the JR line was allospecific for HLA-B7. These CTL lines killed specific target cells at an effector-to-target ratio of 0.4 (VE) or 0.08 (JR). All of the cells, which grow in suspension, rosetted with sheep erythrocytes and reacted with an antiserum specific for human T cells. The CTL line VE was used to raise rabbit antisera that immunoprecipitated two specific polypeptides (78,000 and 33,000 daltons) from labeled membranes of these CTL lines.

Animals↗
PubMedDate not supplied

Lymphokine-activated killer cell generation and recovery. Comparison of an automated cell processing device and a manual procedure.

This report describes the separation or processing of leukapheresis-derived peripheral blood mononuclear cells, their culture in the presence of interleukin-2 (IL-2) to generate lymphokine-activated killer (LAK) cells, and the harvest of LAK cells using the Du Pont SteriCell processor and culture containers. The report compares this automated closed system to the standard National Cancer Institute (NCI) manual protocols. Lymphocytes (approximately 2.0 x 10(10)) were harvested on the Cobe 2997 blood cell separator for each set of experiments. A set of experiments using automated Ficoll-Isopaque (FI) separation and a set of experiments using an automated wash for platelet reduction were compared to the respective manual methods. SteriCell culture containers were compared to roller bottles, and automated harvest was compared to manual harvest. The cytolytic activity of LAK cells was assayed against that of Daudi cells in a standard 51Cr release assay. The cytolytic activity of LAK cells prepared by a manual method and by the automated SteriCell method are equivalent. The manual and SteriCell FI separation procedures were substantially equivalent. The SteriCell harvest method gave higher recoveries than the manual harvest method (p = 0.04 and p = 0.07), but the overall recovery of LAK cells was not significantly different. Differential counts on the products were similar. Platelet reduction by the SteriCell automated wash procedure was greater than that by the manual procedure (p = 0.05). The SteriCell system represents an acceptable alternative to the manual methods for LAK cell generation and recovery.

Adult↗
PubMedDate not supplied

Agranulocytosis and hemolytic anemia in patients with renal cell cancer treated with interleukin-12.

Interleukin-12 (IL-12) is a cytokine with effects on immune function and hematopoiesis. In this article, the authors describe two patients with renal cell cancer in whom grade 4 neutropenia and grade 3 hemolytic anemia developed, respectively, during treatment with twice-weekly intravenous recombinant human interleukin-12 (rhIL-12) during a phase 1 trial. The severe neutropenia was associated with bone marrow agranulocytosis and a preponderance of large granular lymphocytes in the peripheral blood, whereas the hemolytic anemia was negative for the Coombs test and associated with splenomegaly. The agranulocytosis and hemolytic anemia persisted after the rhIL-12 was stopped, but both subsequently responded to treatment with cyclophosphamide. steroids, or both. These findings indicate that rhIL-12 can induce unique hematologic toxic effects that can be reversed with immunosuppressive drugs.

Aged↗
PubMedDate not supplied

Differential effects of hydroxamate inhibitors on PMA and ligand-induced L-Selectin down-modulation: role of membrane proximal and cytoplasmic domains.

L-selectin is down-modulated from the cell surface in response to leukocyte activation or cross-linking with ligand mimetics such as anti-L-selectin antibodies or sulfatides. The down-modulation induced upon cellular activation with PMA is due to proteolytic shedding mediated by a cysteine metalloprotease, presumably the TNF-alpha converting enzyme (TALE), and is susceptible to inhibitors of the hydroxamate class. To determine if cross-linking-induced down-modulation of L-selectin is similarly affected by these inhibitors, we exposed Jurkat T cells to the anti-L-selectin antibody Dreg 200 or sulfatides in the presence of the hydroxamate TNF-alpha protease inhibitor (TAPI). TAPI completely inhibited PMA-induced downmodulation but had no effect on that induced by the anti-L-selectin antibody or sulfatides. The downmodulation induced by an anti-TCR antibody (WT31) was only partially inhibited by TAPI. An L-selectin mutant lacking the putative TACE cleavage site and resistant to PMA-induced shedding (321del.9) was expressed in L1.2 pre-B cells. Like WT L-selectin, this mutant was down-modulated when exposed to sulfatides, or Dreg 200 and this down-modulation was unaffected by TAPI. An L-selectin cytoplasmic tail deletion mutant (344del.15) expressed in L1.2 pre-B cells was down-modulated by PMA or sulfatides, but not Dreg 200. Electrophoretic analysis of L-selectin immunoprecipitated from the supernatants of Jurkat cells treated with either sulfatides or D200 revealed a proteolytic fragment of the same size as that released from the cell surface in response to PMA. Our data indicate that the down regulation of L-selectin in response to cross-linking by ligands or TCR engagement may be mechanistically distinct from that induced by PMA. Furthermore, our results with the 344del.15 mutant suggest that the down-modulation of Lselectin induced by certain sulfated carbohydrate ligands may be initiated through surface receptors other than L-selectin itself. The abbreviations used in this paper are: TAPI, TNF-alpha protease inhibitor; TACE, TNF-alpha converting enzyme; PMA, 4(3-phorbol 12-myristate 13-acetate.

Journal Article↗
PubMedDate not supplied

Effects of human interleukin-1 on natural killer cell activity: is fever a host defense mechanism for tumor killing?

Interleukin-1 (IL-1) represents a family of polypeptides with a wide range of biological activities. cDNA from two gene products has been cloned; there are probably more. The human IL-1 family plays an important role in the pathogenesis of many diseases and functions as a key mediator of host response to various infectious, inflammatory, neoplastic, and immunologic challenges. Recombinant mouse (pI 5) and recombinant human (pI 7) IL-1s are being used to confirm the multiple biological properties of IL-1s. Some IL-1 biological activities seem to be involved with mechanisms of host tumor killing. Incubating purified or recombinant human IL-1 with human peripheral blood mononuclear cells in the presence of IL-2 or interferon-alpha results in a synergistic enhancement of certain tumor cells. More recent results indicate that IL-1 exhibits direct cytotoxicity for tumor cells in vitro. The peripheral blood mononuclear cells of patients with tumors demonstrate decreased production of IL-1 when challenged with endotoxin and show a comparable decrease in natural killer activity; adding exogenous IL-1 reverses this defect in these patients. However, induction of hepatic acute-phase proteins such as serum amyloid A serves as a negative feedback since the amyloid protein suppresses natural killer activity. Moreover, natural killer cell activity in the presence of IL-1 or interferon-alpha is suppressed by incubating temperatures of 39 degrees C. This effect is not reversed by inhibitors of prostaglandin synthesis. IL-1 is clearly important to host defense against malignancy, but some aspects of IL-1 biology seem to exert a contrary influence.

Cytotoxicity, Immunologic↗