Lysis of autologous human macrophages by lymphokine-activated killer cells: interaction of effector cell and target cell conjugates analyzed by scanning electron microscopy.

Lymphokine (i.e., interleukin 2; IL-2)-activated killer (LAK) cells derived from normal human blood are known to destroy human tumor target cells. Accordingly, immunotherapy modalities using IL-2, either alone or in combination with LAK cells, have been evaluated for eradicating metastatic cancer. In studies conducted to characterize receptors on LAK cell membrane ultrastructures, we observed that LAK cells kill autologous human monocyte-derived macrophages (M phi). In these experiments, peripheral blood mononuclear cells of a healthy adult donor were cultured to generate LAK cells and autologous non-adherent M phi. Thereafter, conjugates were prepared by incubating for 3 h autologous populations of LAK cells and M phi. Examination of the conjugates by scanning electron microscopy (SEM) identified LAK cell-mediated killing of M phi. Moreover, SEM analysis of the LAK cell membrane architecture identified microvilli-like ultrastructures that provided a physical bridge that joined together the LAK cell and M phi. The immunological mechanism(s) underling LAK cell killing of autologous M phi is not known; nevertheless, these conjugates will provide a useful model to study membrane receptors on ultrastructures that mediate the initial stages of cytolysis that include target cell recognition and cell-to-cell adhesion. The results of our observations and the findings of other investigators who have also demonstrated LAK cell killing of autologous normal human leukocytes are discussed in the context of the association of IL-2 and IL-2-activated killer cells with side effects observed in ongoing clinical trials and with autoimmune disorders.

Scanning electron microscopic study of immunogold-labeled human leukocytes.

For many years critical point drying (CPD) has been the method of choice for preparing cells for scanning electron microscopy (SEM). Described herein is a simple, efficient, inexpensive, reproducible, and safe procedure using Peldri II, a proprietary fluorocarbon compound that is solid at room temperature and a liquid above 25 degrees C, as a sublimation dehydrant for processing specimens for SEM. The utility of Peldri II was demonstrated in studies using leukocytes from the blood of healthy donors and patients with leukemia as well as from long-term lymphoblastoid cell lines. The application of the proposed Peldri II procedure was further documented in SEM studies in which the expression and distribution of the interleukin-2 receptor (IL-2R) on leukocyte surface membranes was imaged using colloidal gold-labeled antibodies (i.e., immunogold). When compared with current SEM preparation procedures using CPD, Peldri II is a useful alternative that is thought to offer several important advantages.

Long-term cultivation of functional human macrophages in Teflon dishes with serum-free media.

Reported herein are the results of studies demonstrating the utility of a chemically defined, serum-free medium designated as AIM-V (GIBCO) for the long-term (greater than 2 weeks) cultivation of functionally-defined human macrophages. The AIM-V medium is a mixture of HEPES-buffered Dulbecco's Modified Eagle Medium and Ham's Nutrient Mixture F12 that had been supplemented with purified human albumin, transferrin, insulin, and a proprietary mixture of purified factors. Nonadherent macrophages for serial studies were generated in petri dishes with a Teflon liner that had been seeded with a heterogeneous population of peripheral blood mononuclear cells of healthy human adults. For comparison, cultures were initiated with serum-free medium AIM-V and medium supplemented with AB/Rh+ serum or freshly collected autologous serum. Viability was defined by trypan blue dye, glass adherence, and phagocytosis of yeast. Macrophages were characterized by light microscopy, cytochemistry, and phenotypic analysis. Ultrastructural morphology was defined by scanning electron microscopy. These studies demonstrate that functionally defined human macrophages can be sustained in long-term culture with the use of serum-free medium that has not been augmented with mitogenic stimulants, growth-promoting lymphokines/monokines, or differentiation-inducing agents. Serum-free medium AIM-V, which has been approved for generating lymphokine, (i.e., interleukin-2; IL-2)-activated killer cells (LAK) for IL-2/LAK adoptive immunotherapy modalities, may also prove useful for studies defining and isolating regulatory proteins produced by activated monocytes and macrophages and for generating cytolytic macrophages for different antitumor regimens.

Identification of the interleukin-2 receptor (IL-2R) on human leukemic T cells using colloidal gold and scanning electron microscopy.

Results of studies demonstrating the identification of the interleukin-2 receptor (IL-2R; i.e., anti-Tac) on the membrane ultra-structure of human leukemic T cells with an antibody carrying an electron dense colloidal gold microsphere (e.g., immunogold) that was visualized using a scanning electron microscope (SEM) are reported. Our IL-2R model system employed HTLV-1 retrovirus-infected lymphoblastoid cells of the long-term human leukemic T cell line HUT-102B2. The presence of the IL-2R on these cells was defined using a double antibody procedure that employed as the primary antibody a purified mouse monoclonal anti-Leu-IL-2R antibody (mIgGlk, anti-Tac, CD25), and used as the secondary antibody a goat anti-mouse IgG (gamma-chain specific) antibody that had been covalently bonded to a 40 nm colloidal gold particle. More than 95% of the HUT-102B2 were IL-2R+, and there was a uniform distribution of the IL-2R over the surface of the cells. Corresponding controls were employed in all examinations and included IL-2R- Jurkat human leukemic T cells and isotype identical immunoglobulins. The primary and secondary antibody reagents contained whole human serum and bovine serum albumin, and there was no evidence of the non-specific binding of these antibodies. These studies are the first to demonstrate the presence of a lymphokine receptor on the surface architecture of a cell. We anticipate no difficulty in applying the immunogold/SEM technology to define both normal and malignant cell membrane receptors for other cytokines.

Activation and long-term proliferation of human cord blood T cells with human recombinant interleukin-2.

We report here the results of in vitro studies that demonstrate that purified human recombinant interleukin-2 (hrIL-2) is a potent stimulant for freshly isolated cord blood lymphocytes of healthy full-term infants. Different culture parameters were studied to define the optimal conditions for eliciting maximal levels of activation. Highest levels of hrIL-2-induced TdR[3H] uptake were recorded on day 7 for cultures containing 100 U hrIL-2/ml. Results of comparative studies demonstrated that the reactivity of cord blood lymphocytes to hrIL-2 was equal to, if not greater than, that of healthy adult lymphocytes. Cord blood cells that had been activated with hrIL-2 could be propagated in long-term (greater than 30 days) cultures as hrIL-2-dependent lines, and these lines could be initiated with a high degree of success. Phenotypic analysis was performed using different monoclonal antibodies and cytofluorometry, and studies characterizing cells of the long-term lines have shown that they consisted of a heterogenous population of T4 helper and T8 cytotoxic/suppressor cells; in some instances, natural killer (NK) cells were also present. Other experiments demonstrated that hrIL-2-activated and hrIL-2-propagated T cells expressed the IL-2 receptor (IL-2R; defined by monoclonal antibody anti-Tac) and the number of IL-2-R-positive cells could be increased two-fold or more by exposing the cells to a phorbol ester. This report provides additional information to support the hypothesis that hrIL-2 not only sustains T cell proliferation (i.e., second signal) but also induces T cell activation (i.e., first signal).(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of human recombinant tumor necrosis factor on the growth of different human and mouse long-term hematopoietic cell lines.

Previous studies have demonstrated that one of the most salient features of tumor necrosis factor (TNF) is its ability to induce tumor necrosis in vivo, and the specificity of its cytotoxic/cytostatic activity for tumor cells has been demonstrated in in vitro studies in which this lymphokine has been shown to kill cultured cells of malignant lines and to have no effect on cells of normal diploid lines. Studies described herein defined the effect of highly purified human recombinant TNF on cells of 34 different human and murine hematopoietic cell lines, particularly human leukemic T and B cells of long-term lymphoblastoid cultures. Results of these studies demonstrated that TNF at concentrations of 3,600 U/ml had no significant effect on the growth of these cells as defined by cytotoxicity, measured with the use of the trypan blue dye-exclusion assay and as defined by cytostasis, assayed by the enumeration of cells and the uptake of [3H]-thymidine and -uridine. In contrast, positive control cultures of TNF-sensitive cells from a murine tumor (L-M/clone L-929, connective tissue) displayed at 50% (LD50) reduction in growth by TNF at approximately 5 U/ml. Likewise, human tumors (MCF-7, breast, and HT-29, colon) were also highly sensitive (LD50 less than 100 U/ml). These studies demonstrate that T and B cells of lymphoblastoid lines as well as cells of other hematopoietic lines display little or no sensitivity to TNF.

Isolation of mouse T-cell lymphoma lines from different long-term interleukin 2-dependent cultures.

A number of different biological properties have been ascribed to the hormone-like protein interleukin 2 (IL-2). However, the most salient feature of this lymphokine is its ability to sustain the long-term proliferation of T-cells from humans and mice. Reported herein are the results of studies demonstrating the isolation of growth factor-independent cell lines from the long-term IL-2-dependent murine T-cell line CTLL-2 that is used frequently as the source of target cells in IL-2 bioassays. Sustained log-phase growth of these T-cells in vitro has been achieved using Petri dishes of polymethylpentene; growth could not be sustained in similar dishes of glass, untreated polystyrene, polystyrene that had been treated for cell culture, or polycarbonate. The IL-2-independent line grew as a T-cell lymphoma when injected i.p. into pristane-treated, but not untreated, syngeneic C57BL/6 mice. In contrast, cells from the IL-2 parental line CTLL-2 did not grow in vivo. Characterization of the IL-2-independent lines propagated in vitro (denoted as line CEC) or in vivo (denoted as line CEP) demonstrated that they retained their dependency for 2-mercaptoethanol and expressed phenotypic profiles of their parental line CTLL-2 (Thy 1.2+, Lyt-1-; Lyt-2-). Isolation of an IL-2-independent T-cell lymphoma from a CTLL-2 line obtained from another investigator using a protocol that has proven reproducible under carefully controlled laboratory conditions and defined phenotypic traits of the syngeneic T-cell isolates provided evidence that the tumors were not a cross-culture contaminant arising as a result of a laboratory accident. Moreover, karyotypic analysis using a quinacrine:Hoechst banding technique revealed similar marker chromosomes in the IL-2-dependent and -independent lines. IL-2-independent lines have also been established from the IL-2-dependent murine T-cell line CT-6. Accordingly, the results of these studies suggest that, during prolonged cultivation that has included exposure to crude IL-2 preparations known to contain phorbol ester, possibly viruses, and other contaminants, the IL-2-dependent lines have developed subpopulations that are thought to have undergone malignant transformation of unknown etiology to generate IL-2-independent murine T-cell lymphomas that can be passaged repetitively either in vitro or in vivo.

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.