Edwin W. Ades

Antigen presentation by a continuous human microvascular endothelial cell line, HMEC-1, to human T cells

Endothelial cells line the vessels and lymphatics of the body, acting as a barrier between the blood and extravascular tissue. These cells are, therefore, in a prime position to play a role in lymphocyte activation. Indeed, it has been shown that primary endothelial cells in culture are capable of presenting particulate and soluble antigens to T cells and that this response is not dependent on macrophages. Recently, we developed an immortalized line of human microvascular endothelial cells, CDC/EU.HMEC-1 (HMEC-1). This endothelial line has the advantage not only of being devoid of contaminating cells but also of being a continuous cell line and therefore not subject to a restricted number of useful passages. The focus of this study was to determine whether HMEC-1 cells (like primary endothelial cells) could present antigen to T cells in the absence of macrophages. We demonstrate that a cloned and purified endothelial cell line can independently provide all the necessary signals for antigen-specific T-cell activation.

Use of a Human Microvascular Endothelial Cell Line as a Model System to Evaluate Cholesterol Uptake

CDC/EU.HMEC-1 (HMEC-1) cells provide a reliable source of human microvascular endothelial cells free of mycoplasma and viral infection. This cell line has potential for use in the further study of the endothelial cell modification of low-density lipoproteins and for anticholesterol drug evaluation assays. HMEC-1 cells will fill a gap that is present for in vitro investigations of cholesterol metabolism in conjunction with previously established hepatic, monocytic, or macrophage cell lines. This paper presents a simple assay that demonstrates a linear uptake of tritiated cholesterol by he HMEC-1 cells and shows that the cellular cholesterol load can be regulated using anticholesterol drugs.

Immune Responses in Humans while Receiving Adoptive Immunotherapy with Recombinant Interleukin-2 and Lymphokine-Activated Killer Cells: Acute Anergy to Mitogens and Recall Antigens

In this study we evaluated the in vitro immunologic responses of 11 patients receiving immunotherapy with either interleukin-2 (IL-2, 3 X 10(6) units/m2) or IL-2 plus lymphokine-activated killer cells (LAK) over a 30-day period. Blastogenic responses to mitogens or recall antigens were found to significantly decrease during therapy. Pokeweed mitogen immunoglobulin (Ig) production decreased significantly in 7 patients or showed no change. In vivo skin tests for cell-mediated immunity were also performed and the average number of positive responses before IL-2/LAK therapy decreased to no positive responses (day 29 of therapy). Experiments were performed to determine whether decreased responses were a result of active suppression or a dilution effect by immature/mature cells that cannot respond to mitogen or antigen. Pre-therapy cells were mixed with fresh autologous (anergic) cells from day 29 of therapy in varying ratios. Blastogenesis and Ig production was measured. Our findings demonstrate that the decreased immune response observed during and after therapy is a result not of active immunosuppression but rather of a dilution effect by cells with immune dysfunction. We conclude that: (1) in vitro blastogenesis or Ig production tests using mitogens or antigens for patients undergoing IL-2 immunotherapy have no predictive value, and (2) the immune cells that are present are refractory.

Studies of Adenovirus Subtypes and Down-Regulation of HLA Class I Expression: Correlations to Natural-Killer-Mediated Cytolysis

Natural killer cell (NK) cytotoxicity historically has been accepted to be unrelated to major histocompatibility complex (MHC) expression. However, recent studies have indicated that a decrease in MHC antigen expression leads to a concomitant increase in NK cytotoxicity. We have, therefore, studied the alteration of HLA class I expression by 6 types of adenovirus in the human cell line HEp-2. We conclude that for the 6 types of adenovirus tested, HLA class I expression and NK cytotoxicity are not interrelated.

Mammalian tissue culture growth, viral replication, and cultivation using serum replacement factor.

Abstract Variability, cost, and availability of fetal bovine serum are in question; thus, we examined whether using a serum replacement factor in multiple mammalian tissue cultures could support not only cell growth, but also viral replication, expression, and retention of phenotypic markers. By using a serum replacement in defined media, we demonstrated multipassage cell growth of several different cell lines and viral cultivation and replication equivalent to fetal bovine serum. Moreover, by supplementing media with a serum replacement factor we observed at least a 28% financial savings.

Suppression of Human Immunoglobulin Synthesis by Interleukin-4 in Tandem with Interleukin-2 through Large Granular Lymphocytes

Recent studies have shown that interleukin (IL)-4 can affect secretion of immunoglobulins (Igs) or activation of cytotoxic cells by IL-2, while other studies have shown that natural killer (NK) cells/large granular lymphocytes (LGLs) can also affect Ig synthesis. Therefore, we examined the effect of IL-4 with and without IL-2 or human NK/LGLs on pokeweed mitogen (PWM)-stimulated production of IgM and IgG. We found that when IL-4 and/or IL-2 were incubated with peripheral blood lymphocytes and PWM for 7 days and an enzyme-linked immunosorbent assay was run to measure Ig synthesis, IL-4 with IL-2 caused a greater suppression of Ig synthesis than either cytokine alone. A further experiment was done to determine the effect IL-4 and IL-2 would have on LGL suppression of Ig synthesis. IL-4 and IL-2 alone and in combination, when added to LGL, caused the LGL to suppress Ig synthesis to a greater extent than alone. We conclude that IL-4 acts on NK/LGLs separately and jointly with IL-2, to suppress Ig synthesis (IgM and IgG).

Potentiation of Human Natural Killer Cell Activity by Recombinant Interleukin-2 towards Multidrug-Resistant Human Epidermoid Carcinoma

Relatively little is understood about the antigen recognition and target structures used by natural killer (NK) cells. The purpose of this study was to analyze the relationship of a multidrug-resistant cell line expressing the P-glycoprotein (GP170 surface glycoprotein) derived from the parent non-drug-resistant malignant tumor cell line for susceptibility to lysis by either NK or lymphokine-activated killer cells. Our results demonstrate no significant difference in NK activity against either the non-drug-resistant or drug-resistant malignant cell line; Interleukin-2 induces a significant increase in cytolytic activity toward the multidrug-resistant cell line. These data suggest that malignant cells refractory to treatment or occurring after successful treatment are susceptible to immunotherapeutic intervention.

Immunologic effects of interleukin-2 adoptive immunotherapy in humans : acute in vitro anergy, in vivo antibody response to tetanus

In a previous study we evaluated the in vitro immunologic responses of 14 patients receiving immunotherapy with either interleukin-2 (IL-2; 3 x 10(6) units/m2) or IL-2 plus lymphokine-activated killer (LAK) cells over a 45-day period. Blastogenic responses to mitogens or antigens were found to significantly decrease. Pokeweed mitogen immunoglobulin production decreased or showed no change. Multitest skin test response decreased during and after therapy. We concluded that, although natural killer and LAK activity are enhanced during therapy, in vitro blastogenic or immunoglobulin tests using mitogens or antigens for patients undergoing IL-2 immunotherapy have no predictive values and are depressed. In this study, we provide information that patients while receiving IL-2/interferon-alpha immunotherapy demonstrate as in the previous study in vitro reduced immunologic responses by at least 60%; however, in vivo, they had a normal immunoglobulin response to a tetanus booster. The disparity in results (in vitro versus in vivo) is unexplainable. Further analysis of other in vitro and/or in vivo tests is required to determine the effect IL-2 immunotherapy may have on the immune response status.

Down-Regulation of lnterleukin-2 Receptor Expression on Natural Killer Cells/Large Granular Lymphocytes by lnterleukin-4

It has recently been demonstrated that IL-4 inhibits IL-2 receptor expression on T cells. Studies have also shown that IL-4 can inhibit IL-2-induced natural killer cell (NK) cytotoxicity, and that IL-4 in combination with IL-2 and large granular lymphocyte (NK/LGL) cells suppresses Ig synthesis. Therefore, we examine whether IL-2 receptor expression on NK/LGL cells is affected with or without IL-4, using fluorescent receptor analysis. Our results demonstrate that IL-4 inhibits/down-regulates the expression of IL-2 receptors on either phyto-hemagglutinin or IL-2-stimulated NK/LGL cells.