Peter Ralph

Human leukemic models of myelomonocytic development: a review of the HL-60 and U937 cell lines.

The human leukemic myeloblast HL‐60 and monoblast U937 cell lines have made important contributions to the disciplines of cancer, hematology, and immunology. As sources of leukemic cells, they have been used for the study of neoplasia and therapeutics. As sources of hemic cells, they have been used for biochemical and biological analysis of regulation and differentiation in myelopoiesis. When stimulated with immunomodulatory factors, the cell lines develop properties of host‐defense effector cells. They are also a source of cytokines that affect other cell types.

Interleukin-2 correction of defectivein vitro T-cell mitogenesis in patients with common varied immunodeficiency

We studied the ability of phytohemagglutinin (PHA) and two anti-T-cell monoclonal antibodies, OKT3 and Pan T2, to induce interleukin-2 (IL2) production and proliferation in peripheral blood lymphocytes (PBL) from 14 patients with combined varied immunodeficiency (CVI). The median values of endogenous IL2 produced by mitogen-stimulated PBL was significantly lower in patients than controls irrespective of the mitogen used. The patients, taken as a group, had a significantly decreasedin vitro PBL response to mitogen stimulation when compared to controls. With the addition of a highly purified human IL2 preparation, the proliferative response in the majority of patients was significantly improved with all mitogens. Three patient groups could be distinguished: Group A (3/14) had full restoration of proliferative response with the addition of IL2, Group B (5/14) had partial restoration, and Group C (6/14) had no significant response. The monoclonal antibody, Pan T2, recognized a T-cell proliferative defect in 5 of 14 patients which neither PHA nor OKT3 recognized. This was not significantly corrected by the addition of IL2. This T-cell proliferative defect correlated with the lack of B-cell proliferation and immunoglobulin production in response to B-cell mitogens in three-fourths of the patients assayed. These data show that CVI patients are a heterogeneous group but have in common a decreasedin vitro production of IL2 resulting in a proliferative defect which is correctable at least in part,in vitro, in the majority by the addition of purified IL2.

Induction of IgG production in human B lymphoblastoid cell lines with normal human T cells.

THYMUS-DERIVED (T) lymphocytes play a critical part in the induction of B lymphocytes to antibody production1, especially for conversion of IgM to IgG response2. In humans, the presence of T lymphocytes is also essential for the induction of IgM or IgG production by pokeweed mitogen (PWM) stimulated B lymphocytes3,4. In many experiments it has been shown that antigen-specific5 or nonspecific6–8 soluble factors from T cells, together with antigen or other inducers, for example, anti-immunoglobulin antibody, acting on B-cell surface receptors9, can also provide the stimulus for Ig production in B cells. However, the chemical nature of a B-cell acceptor for the T–effector molecule, the biochemical events responsible for the differentiation of B cells to Ig-producing cells, and the mechanism of the switch of transcription from μ chain to γ chain genes under the influence of T cells remain largely unknown. Heterogeneity of B-cell population in spleen, lymph node and blood has hindered the molecular analysis of immune phenomena. In this situation, B lymphoblastoid cell lines may be useful models for such analysis, since they may be arrested at certain phases of their differentiative history and if influenced by T cells or T-cell factors might permit incisive analysis of induction and switching of gene action at the molecular level. We report here the induction of IgG production or enhancement of IgM secretion in several human B lymphoblastoid cell lines with the help of normal T cells.

Tumor promotor phorbol myristic acetate is a T-cell-dependent inducer of immunoglobulin secretion in human lymphocytes

Abstract Human peripheral blood mononuclear cells were induced by 10 to 200 ng/ml phorbol myristic acetate (PMA) to secrete immunoglobulin (Ig). Using protein A-RBC plaque formation to detect secreting cells (PFC), Day 5 and Day 7 PFC induced by PMA were 0.2 to 1.4% of initial numbers of cultured cells. In parallel cultures, pokeweed mitogen (PWM) induced PFC corresponding to 1–3% and 0.6–8% of initial cell numbers, respectively. Suppressor cells for Ig secretion purifying with T (E-rosetting) cells were found in most donors. These were sensitive to mitomycin C. In some donors, PFC responses could only be detected after removal of suppressor cells. By double separation of T cells from B cells, the PMA response was shown to be dependent on T helper cells, similarly to PWM. This is in contrast to a 20-fold stimulation of Ig secretion in some human B culture lines by PMA alone. PMA is thus a T-dependent polyclonal activator for resting, blood B lymphocytes, and a direct stimulator of Ig production in certain dividing lymphoblasts represented by B-cell lines.

IgM and IgG secretion in human B-cell lines regulated by B-cell-inducing factors (BIF) and phorbol ester

Human B-cell lines were screened for stimulation of immunoglobulin production by incubation with lymphokine (LK) or tumor promoter, phorbol myristic acetate (PMA). One group of lines had essentially no immunoglobulin-secreting cells (ISC) under any condition (less than 0.01%), detected by a reverse plaque assay. Another group of lines had high levels of ISC (greater than 5%) which was not increased substantially by inducing agents. In a third group of IgM and IgG lines, there were intermediate levels of ISC which could be increased by LK, PMA or both agents. No evidence for isotype switching in a number of stimulated IgM and IgG cell lines was detected. Clone SKW6.4 of an IgM line was highly responsive to a B-cell-inducing factor (BIF) in LK. BIF for SKW6.4 and IgG line ARH-77 was weakly binding to DEAE cellulose, about 20,000 mol. wt., and separable from IL-2 by blue agarose chromatography. IL-2 did not stimulate secretion in SKW6.4 with or without purified BIF. In Clone SKW6.4, BIF stimulated ISC per recovered cell up to 30-fold by day 1 of culture, and these plateau levels of about 6% ISC were maintained for longer than 4 days. Treatment of cells with BIF for less than 1 day was sufficient to produce maximum effect on this clone for the succeeding 4 days. Cells stimulated with BIF and then subcultured at day 3 without BIF showed ISC numbers increasing but at a slower rate than the total population, suggesting that the induced differentiation state is long-lived (half-life of % ISC greater than 6 days) and that ISC produce some daughter ISC.(ABSTRACT TRUNCATED AT 250 WORDS)

Induction of human immunoglobulin secretion: II. T lymphocyte dependency and radiosensitivity of T-cell help for induction of B-cell differentiation by staphylococcus aureus strain cowan I

Abstract The induction of peripheral blood B lymphocytes to mature to immunoglobulin-secreting cells (ISC) when stimulated by Staphylococcus aureus strain Cowan I was found to be T helper cell-dependent ( J. Immunol. 127 , 1044, 1981). The nature of T help was studied in B- and T-cell separation and reconstitution experiments. T helper cells for Cowan I were very radiosensitive (D 37 37 > 2000 rad). PWM synergized with Cowan I in induction of ISC, and helper T cells for dual stimulation were also radioresistant. The ratio of T to B cells was found to be critical in judging reactivity of donors for both PWM and Cowan I. T cells stimulated with PWM, but not Cowan I, produced T cell-replacing factors essential for Cowan I-induced maturation of B cells. Irradiation of T cells prior to PWM stimulation increased the level of such helper factors. Poor responders to Cowan I, as judged by mononuclear cell cultures, had apparently few helpers for the bacterial stimulant, compared to high responders. Cowan I helper T-cell activity did not appear to be due to protein A leaking from the bacteria and stimulating T cells. In all these experiments, induction of ISC by Cowan I was completely dependent on T cells or factor, providing a good model for investigation of B-cell differentiation regulated by a unique subset of radiosensitive T helper cells.

Rescue of IgM, IgG, and IgA production in common varied immunodeficiency by T cell-independent stimulation with Epstein-Barr virus

We previously defined three categories of B-cell defects in common varied immunodeficiency (CVI): failure to produce IgG and IgA in response to T cell-dependent (TD) stimulation byStaphylococcus bacteria (Sac) plus pokeweed mitogen or B-cell inducing factor (BIF), failure to produce any immunoglobulin, and failure of Sac-induced proflieration and differentiation. The present study includes the responses of 22 CVI patients to T cell-independent (TI) stimulation by Epstein-Barr virus (EBV). In the majority of patients, EBV-stimulated B cells showed normal proliferation and IgM production. In addition, IgG and IgA production was in the range of that for EBV-stimulated normal cells in many patients. Among 11 patients with no TD production of immunoglobulin of any isotype, two showed normal IgM secretion in response to EBV and five others had significant but subnormal responses. Four patients never had humoral responses despite repeated testing and removal of potentially suppressing T cells and monocytes. Concanavalin A stimulation of the T cells from all the patients tested resulted in the production of B-cell inducing factor at higher levels than for normal donor T cells, as assayed on normal Sac-stimulated B cells. These results show that many cases of B-cell defects in CVI patients involving TD production of IgM, switching to TD production of IgG and IgA, and mitogen responses to Sac are not absolute defects. The B cells will respond normally to some stimuli.

T-Cell Growth Factor (Interleukin 2) and Terminal Transferase Activity in Human Leukemias and Lymphoblastic Cell Lines*

SummaryMononuclear blood cells from patients with different types of leukemia, and from controls as well as cells from established lymphoblastic cell lines were analyzed with respect to terminal transferase (TdT) activity and T-cell growth factor (TCGF; Interleukin 2, IL-2), to determine the significance of TCGF production and response as functional markers for human leukemias.The data obtained so far suggest that the aberrant proliferation and lack of maturation observed in these leukemias may be associated with or be the result of a break-down in cellular-mediated control of proliferation.

Activation of cyclic AMP-dependent protein kinase activity during LPS stimulation of macrophage tumor cell line, J774.1.

Abstract Stimulation of J774.1 macrophage cell line with lipopolysaccharide (LPS)- or 8Br-cyclic AMP induced phosphorylation of nonhistone nuclear proteins (NHP) particularly of size range 35,000 daltons. In vitro phosphorylation of nuclei isolated from LPS- or 8Br-cyclic AMP-stimulated cells also showed an increased incorporation of 32 P into NHP indicating an activation of a NHP-specific protein kinase in nuclei. Incubation of normal nuclei with cytoplasmic extracts from LPS- or 8Br-cyclic AMP-stimulated cells increased NHP-specific phosphorylation in nuclei. Incubation of normal nuclei with normal cytoplasm in the presence of 1 × 10 −8 M cyclic AMP also induced an increased phosphorylation of NHP, suggesting that the LPS-induced increase of intracellular cyclic AMP activates a cyclic-AMP-dependent NHP-specific protein kinase in the cytoplasm, which moves into the nucleus to phosphorylate NHP. The increased phosphorylation of NHP proceeded in advance of the secretion of T-cell activating factor(s) and the optimum concentrations of LPS- or 8Br-cyclic AMP for the maximal induction of NHP-specific protein kinase activity were similar to those required for the induction of T-cell activating factor(s) in J774.1. These experiments suggest the intimate involvement of NHP phosphorylation in the induction processes of macrophage factor production.

Augmentation of macrophage antibody-dependent killing of tumor targets by microtubule inhibitors.

Abstract Antibody-dependent cellular cytotoxicity (ADCC) to tumor targets was studied using murine resident peritoneal macrophages and a macrophage cell line RAW264.10A, both having low inherent cytolytic activity. The target was 125 I-labeled pre-B lymphoma 18-8. Pretreatment of both macrophage populations with 0.5 – 2 μ M concentrations of the microtubule-stabilizing drug taxol greatly increased their antibody-dependent cytotoxicity with no stimulation of nonspecific killing. Taxol present only during the 18-hr cytolytic assays had no effect on target killing. Optimal killing activity was obtained by treating macrophages 2 days with taxol, similar to previously described cytokine stimulation of ADCC. This concentration completely blocked growth of RAW264 cells. Other microtubule inhibitors, lidocaine and colchicine, also augmented peritoneal and cell line macrophage ADCC at cytostatic concentrations. In contrast, the microfilament-disrupting agent, cytochalasin B, caused little or no stimulation of ADCC. These results show that microtubule reformation is not necessary for the development of cytotoxicity. Since microtubule inhibitors block lysosomal discharge, they may stimulate macrophage ADCC by causing accumulation of toxic molecules involved in cytotoxicity.