John L. Pauly

A Simplified Technique for in Vitro Studies of Lymphocyte Reactivity

Summary In vitro studies of lymphocyte reactivity to mitogens, soluble antigens and homologous cells were performed by diluting whole blood with culture medium and using 24-hr labeling with 3H-thymidine. Sensitivity, reproducibility and correlations with delayed skin test responses of the blood donors were superior to those obtained with conventional techniques using lymphocyte concentrates. At appropriate reactive cell densities, supplementation of the culture medium with agents such as fetal calf serum has not been necessary. This simplified technique offers considerable savings in effort, cost and volumes of blood required.

Elevated thymidine phosphorylase activity in the plasma and ascitic fluids of tumor-bearing animals.

Summary Activity of thymidine (TdR) phosphorylase (EC 2.4.2.4; TdR: ortho-phosphate deoxyribosyltransferase) in the plasma of healthy and tumor-bearing mice and rats was evaluated in comparative studies. Increased enzyme activity was recorded in 9 of 11 tumor models tested, and highest values were obtained for mice with lymphoid or myeloid leukemia. Heightened activity of this TdR-degrading enzyme was observed in the plasma of mice bearing three of four solid tumors. Elevated TdR phosphorylase activity was also identified in the peritoneal exudate fluids in each of four groups of animals carrying ascites-producing tumors.

Studies of cultured human T lymphocytes. I. Production of the T cell growth-promoting lymphokine interleukin-2☆☆☆

Described herein is a large-scale procedure that has been successfully employed for producing 62 lots (800-3000 ml) of supernatants containing the T cell growth-promoting factor Interleukin-2 (IL-2). The efficiency of these crude, unconcentrated supernatants was documented in studies in which 70 human long-term (greater than 100 days) IL-2-dependent T cell lines were established from 50 different donors. These included lines initiated from the peripheral blood of healthy subjects (N = 54), blood of children with active acute lymphoblastic leukemia (N = 6) and the thymus of children undergoing surgery to correct congenital heart defects (N = 10). The underlying concept used in constructing this method emphasizes the requirement of the monocyte-derived macrophage and its Interleukin-1 (IL-1) product to mediate IL-2 production by activated T cells. The most salient feature of this technique is the utilization of buffy coat leukocytes that had been pooled from several blood donors and sustained in spinner cultures for several days prior to polyclonal activation with phytohemagglutinin and pooled B cells of established human lymphoblastoid lines.

A method for isolating human lung macrophages and observations of fluorescent phagocytes from the lungs of habitual cigarette smokers

We report the development of a simple, efficient, expedient, and inexpensive procedure for isolating a large and relatively pure population of macrophages (Mphi) from residual (i.e., non-tumor) lung tissue obtained from lung cancer patients undergoing either a lobectomy or pneumonectomy. The proposed technique was founded on observations by fluorescent microscopy of fresh, non-fixed, and non-stained human lung tissue. Examinations of 74 specimens from different patients revealed that most of the Mphi reside as non-adherent cells within the sponge-like lung stroma. Very few Mphi were observed in the lungs of nonsmokers. In contrast, many Mphi were visible in the lungs of habitual smokers. For most specimens from smokers, a few of the Mphi were present as randomly distributed single cells; the majority of the Mphi, however, were in clusters that ranged from a dozen to several hundred cells. The Mphi could be released readily by different mechanical techniques. In the procedure reported herein, pulmonary leukocytes (> 75% Mphi) were dislodged easily from lung tissue with the use of an inexpensive, hand-operated, tissue grinder. The grinder consisted of a glass mortar and Teflon pestle that provided sufficient clearance between the mortar and pestle so as to avert damaging the displaced leukocytes. The leukocytes were then segregated by centrifugation on a density gradient. Further purification was achieved by harvesting Mphi that had been allowed to adhere to serum-coated polystyrene culture dishes (> 90% Mphi). In most experiments, the Mphi yield (approximately 5 x 10(6) Mphi /gr of lung) and Mphi viability (> 85%) were good. A significant advantage of this technique is that it avoids jeopardizing the cells to the hazards associated with enzymes that have been used in techniques employed previously for isolating Mphi from the lung and other organs. Thus, the proposed method provides numerous lung Mphi for detailed studies of their morphology, phenotype, and function. Moreover, lung Mphi were cultured as non-adherent, single cells in a serum- and cytokine-free tissue culture medium for more than 6 weeks. Lung Mphi from habitual smokers displayed a high level of fluorescence that was readily apparent when viewed with a fluorescence microscope that had been configured with either a fluorescein or rhodamine filter. Serial sections of single, living Mphi obtained with the use of a confocal laser scanning microscope revealed that the fluorescence originated from cytoplasmic inclusions. Relative fluorescence intensity was measured by cytometry.(ABSTRACT TRUNCATED AT 400 WORDS)

Tumour-cytolytic human monocyte-derived macrophages : a simple and efficient method for the generation and long-term cultivation as non-adherent cells in a serum-free medium

We report a simple and efficient culture procedure for the generation of tumour-cytolytic human monocyte-derived macrophages (MAC). In this method, normal human peripheral blood mononuclear cells, isolated using a conventional Ficoll-Hypaque density gradient procedure, are cultured as a heterogenous leukocyte population in Teflon or other hydrophobic cultureware, in a commercially available serum-free culture medium (M-SFM) that has been formulated specifically for the cultivation and ex vivo stimulation of human monocytes and MAC, and in the absence of exogenous mitogens, antigens, cytokines or other stimulants. This procedure features a negative-selection technique that takes advantage of the differential survival of blood leukocytes. Using the prescribed in vitro conditions, lymphocytes survived relatively poorly, whereas monocytes differentiated in the absence of exogenous stimulants into mature tumour-cytolytic MAC. The MAC were present as non-adherent, single cells that expressed good viability (greater than 95%) for a prolonged period (greater than 60 days). When compared to conventional procedures for generating MAC, the prescribed technique is thought to offer several important advantages in that it: (a) eliminates the tedious and cumbersome monocyte isolation procedures, thus providing a significant savings not only in time and money but also in eliminating repetitive cell manipulations that have often been associated with damage to monocyte morphology and/or function; (b) reduces the loss of monocyte subsets that are not recovered during specific isolation procedures; (c) facilitates harvesting a single cell, non-adherent suspension of immunocompetent MAC suitable for various examinations including analyses defining MAC morphology, cytochemistry, phenotype and function; and (d) eliminates variability and artifacts associated with different sera that are utilised frequently as medium supplements. The utility of the prescribed method is illustrated by the results of ongoing studies in which scanning electron microscopy and confocal laser scanning microscopy are being used to define MAC function in different immunological reactions, and examples of these observations are presented herein.

Phenotyping of malignant hematopoietic cells

Summary1255 cases of leukemia-lymphoma were tested between 1972 and 1984 by multiple marker analysis. Routine leukemia phenotyping was performed using standard morphological and cytochemical techniques in combination with clinical and histo-pathological information; the main emphasis was put on immunological surface marker analysis using erythrocyte rosette assays, TdT and a large panel of poly- and monoclonal antibody tests. The 1255 cases were divided into these major types and subtypes: 349 cases of ALL and related immature T- and Burkitt-lymphomas (cALL, pre B-ALL, B-ALL and Burkitt-lymphomas, T-ALL and immature, mostly leukemic T-lymphomas, Null-ALL), 454 cases of mature T- and B-cell malignancies (T-CLL, mycosis fungoides, Sezary-syndrome, T-lymphomas, B-CLL, hairy cell leukemia, multiple myeloma, B-lymphomas), 263 cases of acute myeloid leukemias (AML, AMMoL/AMoL), 182 cases of chronic myeloid leukemias (CML in chronic phase, CMoL, CML in blast crisis), 6 cases of erythroleukemia and 1 case of megakaryoblastic leukemia. A simplified classification scheme which has been used in our laboratories is presented. Phenotyping is of diagnostic, prognostic and therapeutic relevance, most evidently for patients with ALL. Routine leukemia phenotyping should be performed with highly standardized techniques and reagents and by combining information from several fields in the multiple marker analysis. New areas of leukemia research might become very useful for the routine procedure of phenotyping.

Studies of Cultured Human T Lymphocytes II. Initiation of Paired T- and B-Cell Lines from Healthy Donors

Abstract We report the sustained cultivation of both B- and T-lymphoblastoid cell lines from randomly selected healthy donors, and the results of studies denning the frequency with which these cell lines can be established. B-cell lines were initiated using the Epstein-Barr virus. Of 52 attempts, 40 B-cell lines (77% success) were obtained from 24 different donors. T-cell lines were started and propagated in long-term (>100 days) cultures using the T-cell growth factor inter-leukin-2 (IL-2). Of 55 attempts, 54 (98%) were successful in initiating IL-2-dependent T-cell lines, and these were derived from 28 healthy adults. Likewise, of 45 attempts, 32 (71%) were successful in producing paired lines in which both the B-cell line and T-cell line were cultivated from a single blood collection (N = 22 donors). Phenotypic profiles of these lines were defined using multiple marker assays, including rosette formation, surface immunoglobulins, cytochemistry, karyotype, as well as xenoantisera and monoclonal antibodies defining different membrane antigens. This work demonstrates the feasibility of propagating paired human B and T lymphoblastoid lines suitable for many comparative immunobiological studies.

Degradation of [3H]thymidine by a pentosyltransferase (EC 2.4.2.4) in the plasma of man and different animals

[3H]Thymidine is degraded by an enzyme (thymidine phosphorylase; EC 2.4.2.4) which we have identified in the plasma of man and some animals. The presence of this enzyme in plasma or sera used to supplement culture media may, under certain experimental conditions, limit the validity of measuring the uptake of radiolabeled thymidine as a means of defining DNA synthesis.

Isolation of interleukin 2 (IL-2) from human and mouse lymphocyte culture supernatants by batch adsorption onto silicic acid

Described herein is a simple, efficient and inexpensive batch adsorption procedure for the isolation and partial purification of the hydrophobic T cell growth-promoting lymphokine interleukin 2 (IL-2) from crude culture supernatants (SN) of freshly isolated human lymphocytes and leukemic T cells of established lines including human Jurkat J6.2, Gibbon ape MLA-144 and mouse EL-4. In this method, IL-2 was isolated by batch adsorption onto microparticulate silicic acid (SA) by stir-mixing the SA with SN (10 mg/ml; 30 min; 37 degrees C). Thereafter, the SA was pelleted by centrifugation and washed twice with phosphate-buffered saline (PBS). The IL-2 was eluted by adding to the pelleted IL-2-binding SA 5 vols. of ethylene glycol (EG; 50%, v/v) in PBS (pH 7.2) with high salt (1.4 M NaCl). The lymphokine-rich concentrate was then dialyzed (6 kDa MWCO) against PBS to remove the EG and low molecular weight growth inhibitors. The application of the proposed procedure was further defined in experiments in which SA was successfully employed for recovering IL-2 from SN of cultures in which the medium had been supplemented with fetal calf (FCS) or human serum to achieve maximal lymphokine production. Also presented are the results of experiments defining the SA adsorption of proteins from whole sera (e.g., FCS, calf, human and horse) and the relative affinity of different purified proteins for this matrix (e.g., bovine serum albumin, human serum albumin, casein hydrolysate, bovine gamma-globulin and bovine beta-lactoglobulin). The proposed SA procedure may prove useful for isolating other hydrophobic immunoregulatory molecules, and a 2-step purification scheme is anticipated in which the SA adsorption procedure will be used as a preparative method preceding reverse phase high performance liquid chromatography and monoclonal antibody affinity chromatography.

A simple screening assay for substance affecting lymphocyte reactivity

A simple method is described for assaying substances modifying lymphocyte reactivity in vitro to mitogens and antigens. This procedure employes whole blood, microtiter plates and an automated cell harvester. The simplicity and expediency afforded by this assay, which requires very little blood, enables the large-scale screening of different test materials which may be of experimental or clinical merit.