Arabella B. Tilden

Phase I trial of the chimeric anti-GD2 monoclonal antibody ch14.18 in patients with malignant melanoma

The chimeric monoclonal anti-GD2 antibody ch14.18 is made up of the variable region of the murine anti-GD2 antibody 14.18 (or its IgG2a switch variant 14G2a) and the constant region of human IgG1k. Ch14.18 mediates antibody dependent cytotoxicity and complement dependent lysis in vitro. In a phase I trial, 13 patients with metastatic melanoma received ch14.18 as a single dose of 5-100 mg. Therapy was associated with an infusion-related abdominal/pelvic pain syndrome, which required intravenous morphine for control. The pharmacokinetics of ch14.18 best fit a two-compartment model with a T1/2 alpha of 24 +/- 1 hr and a T1/2 beta of 181 +/- 73 hr. Eight of 13 patients developed a weak-modest antibody response directed at the variable region of ch14.18. Clinical antitumor responses were not observed at the doses employed in this study. However, patients receiving greater than 45 mg of ch14.18 had antibody detectable on tumor cells analyzed by fluorescent activated cell sorter. Further modification of the therapeutic regime employing larger doses and frequent administration of ch14.18 are planned.

Excessive prostaglandin E2 production by suppressor monocytes in head and neck cancer patients

The proliferative response of peripheral blood mononuclear cells (PBMC) to the mitogens PHA and Con A was significantly depressed in 86% of 45 head and neck cancer patients compared with 44 normal controls. This depression of immune competence was greatest in older patients and in those with more advanced disease stages. The abnormal mitogen responses could be restored toward normal (especially with Con A stimulation) by incubating the cells with either of two prostaglandin synthetase inhibitors (indomethacin or RO-205720). This augmentation of immune response was independent of other factors, including the primary tumor site, disease stage, treatment (surgery, radiation therapy, or chemotherapy) or the patients age or race. The most likely explanation for this depressed level of immunocompetence was an excessive production of PGE2 by suppressor cells. This was confirmed by the finding that PBMC from patients produced more PGE2 than PBMC from normal individuals (8.4 ng/ml vs. 5.2 ng/ml, p = 0.002). This difference was greatest among patients less than 60 years of age whose cultured PBMC produced 91% more PGE2 than controls (p < 0.0007). Virtually all of the PGE2 was produced by a population of monocytes defined by a monoclonal antibody and purified with a fluorescence-activated cell sorter. Patients with epidermoid cancer of the head and neck thus have an abnormality of immunoregulatory monocytes that can contribute significantly to their depression of cellular immunity by elaborating prostaglandin E2. This abnormality could be partially corrected in vitro by incubating their PBMC with a prostaglandin synthetase inhibitor.

Patients with condyloma acuminatum exhibit decreased interleukin-2 and interferon gamma production and depressed natural killer activity

Peripheral blood mononuclear cells were obtained from 20 untreated condyloma acuminatum patients and from an equal number of sex- and age-matched controls and assayed for cell surface antigen expression, natural killer activity, and lymphokine production. Patient peripheral blood mononuclear cells had significantly lower helper-to-suppressor T-cell ratios (Leu3/Leu2) (p<0.05) and significantly higher percentages of Leu 2+ Tac+ cells (activated suppressor/cytotoxic cells) (P<0.05) and Leu 2+ OKM1+ cells (suppressor cells) (P<0.01). Natural killer activity of condyloma acuminatum patients was significantly lower (P<0.05) than that of controls. Production of interleukin-2 and interferon gamma, but not interferon alpha, was significantly (P<0.01) decreased in condyloma acuminatum patients. There was an inverse correlation between thein vitro production of interleukin-2 and interferon gamma and the percentage of Leu 2+ OKM1+ cells (suppressor) (P<0.01). Thus, patients with condyloma acuminatum differ from controls by demonstrating (1) decreased natural killer-cell activity, (2) decreased production of lymphokines which enhance natural killer-cell activity (i.e., interferon gamma and interleukin-2), and (3) an increased proportion of T cells with a suppressor phenotype.

Flow cytometry evaluation of cell-mediated cytotoxicity

A novel flow cytometry method for the evaluation of cell-mediated cytotoxicity is described. This method uses flow cytometry analysis to distinguish target cells from effector cells by differences in volume and light scatter characteristics. Non-viable target cells, following their interaction with effector cells, are determined via propidium iodide (PI) dye exclusion and then expressed as a percentage of the total target cell population. This assay is suitable both for analysis of systems which allow recycling of cytotoxic effector cells (total cell cytotoxicity assays, TCCA), and of systems in which recycling does not occur (single cell cytotoxicity assays, SCCA). Natural killer (NK) cell-mediated cytotoxicity evaluated by flow cytometry is significantly correlated with the standard 51Cr release assay. Flow cytometry can also be used to evaluate the competitive inhibition that certain cell types exert on the cell-mediated killing of NK-sensitive targets. A prerequisite for this assay is that competitor cells and target cells are distinguishable through their volume and light scatter characteristics. Advantages and pitfalls of the flow cytometry method are discussed, in comparison with the 51Cr-release assay.

Potentiation of IL-2-induced T-cell proliferation by retinoids☆

We evaluated the capacity of retinoids to potentiate proliferative responses of murine T-cells to recombinant human interleukin 2 (rIL-2). Concanavalin A (Con A) prestimulated spleen cells responded in a dose-dependent manner to added rIL-2. All-trans-retinoic acid (RA) at 10(-8) M potentiated the proliferative response by fivefold at saturating levels of IL-2. In similar experiments, two closely related retinamides, all-trans-(phenyl)retinamide (PR) and N-(4-hydroxyphenyl)retinamide (4-HPR), also potentiated murine splenocyte rIL-2 responses. Potentiation of IL-2-induced proliferation was dose-responsive to the concentration of added retinoid with peak potentiation occurring at 10(-10) - 10(-8) M in the presence of 10 U/ml rIL-2. Significant potentiation was observed at retinoid concentrations as low as 10(-14) M. Fluorescence flow cytometry of the responding cells revealed that among L3T4+, Lyt-2+ or total T-cells, at 72 h following Con A stimulation, essentially all of the cells expressed IL-2 receptors (IL-2R). This apparently represents near maximum IL-2R expression and treatment of the cells with retinoids did not increase IL-2R expression at that time point. The potentiation of IL-2 responses by retinoids was also observed with IL-2-dependent HT-2 cells, 98% of which were IL-2R positive. HT-2 proliferative responses to rIL-2 were potentiated as much as fourfold by 10(-10) M RA. HT-2 proliferative responses to rIL-2 were potentiated by all three retinoids dose dependently. Significant potentiation was observed with as little as 10(-14) M retinoid. Retinoids in the absence of IL-2 induced no proliferative responses. These data suggest that retinoids can augment the capacity of IL-2 to induce T-cell proliferation using Con A-activated murine splenic T-cell blasts and a long-term-cultured T-cell line.

Isolation and characterization of Leu 7+ germinal-center cells with the T helper-cell phenotype and granular lymphocyte morphology

Leu 7+ cells in germinal centers of lymphoid tissues largely (>90%) coexpress the T helper-cell marker, Leu 3. In this study we have isolated Leu 7+ (Leu 3+) cells from pharyngeal and palatine tonsils and we have analyzed their surface phenotype, morphologic and cytochemical characteristics, and functional properties. All of these features have been compared with those of T helper-cell populations with natural killer (NK)-like characteristics that we have previously described in peripheral blood. Leu 7+ (Leu 3+) cells from tonsil germinal centers display morphological and cytochemical features of granular lymphocytes and express the T3 marker in the absence of Leu 15. Following stimulation with phytohemagglutinin (PHA) or anti-T3, Leu 7+ (Leu 3+) cells express interleukin-2 (IL-2) receptors and proliferate to some extent in response to IL-2. The localization of Leu 7+ (Leu 3+) cells in B-dependent areas of lymphoid tissues suggests that they may play a regulatory role in B-cell proliferation and/or differentiation. Here we show that Leu 7+ (Leu 3+) cells do not produce B-cell growth factor (BCGF) and do not help pokeweed mitogen (PWM)-driven B-cell differentiation. Therefore, Leu 7+ (Leu 3+) germinal-center cells are distinct from “classic” T-helper cells of blood and lymphoid tissues.

Effects of copper-based dental casting alloys on two lymphocyte cell lines and the secretion of interleukin 2 and lgG

In the oral environment, gingival lymphocytes are involved in maintaining the local immune defense of periodontal tissues. The corrosion rates of copper-based dental casting alloys and the accumulation of corrosion products in host gingiva raise concerns about the effects of these corrosion products on immune responses in the oral cavity. The aim of this study ws to investigate the hypothesis that immune function may be altered by copper dental alloy corrosion products. In vitro cell culture studies were used to analyze the effects of three copper-based dental alloys on a T-cell and B-cell line and their secretion of soluble immune mediators (IL-2) and effectors (IgG), respectively. Results of this study revealed that corrosion products released from copper alloys in 24 h have the ability to reduce cellular viability, alter proliferation, and modulate the production of soluble immune mediators. These results support the hypothesis that copper dental ally corrosion products may alter immune responses and thereby contribute to a variety of dental pathological conditions.

Establishment of Tac-negative, interleukin-2-dependent cytotoxic cell lines from large granular lymphocytes (LGL) of patients with expanded LGL populations

Cell lines were established from purified large granular lymphocites (LGL) isolated from the peripheral blood of seven patients with phenotypically homogeneous LGL expansions. LGL were stimulated with phytohemagglutinin (PHA) or recombinant interleukin-2 (rIL-2) and further expandedin vitro in IL-2-containing media. The surface phenotype of LGL, as assessed by monoclonal antibody staining, was T3+ T8+ in five patients, T3− T8− in one, and T3+ T8− in another patient. The cells also expressed Leu 7, Leu 11, and/or OKM 1 markers in various proportions and were identifiable as LGL by their morphological and cytochemical features. The original surface phenotype of the unstimulated LGL was retained in the IL-2-dependent cell lines from each individual patient, i.e., T3+ T8+ cells originated T3+ T8+ cell lines and T3− T8− cells originated T3− T8− cell lines. Other markers, such as Leu 11 and OKM 1, were generally lost in culture. LGL proliferated in response to rIL-2 but did not express detectable IL-2 receptors, even after prolonged periods of culture. All cell lines from each individual patient had the same surface phenotype, and within the single lines, all of the cells expressed the same markers. Cell lines from two patients consistently displayed chromosomal abnormalities. Although different in the two patients, the abnormalities were identical in all of the lines from the same patient and detectable in most of the cells examined, suggesting a clonal origin for the abnormally expanded LGL populations. Freshly isolated LGL did not exert NK activity. However, the IL-2-dependent LGL lines acquired the ability to kill K562 target cells and to produce gamma interferon (γ-IFN). No direct correlation was observed between these two properties.

Lysis of fresh solid tumor targets in the presence of Con A is mediated primarily by Leu 7+ peripheral blood T lymphocytes: Blocking by the anti-CD3 monoclonal antibody and comparison with recombinant interleukin 2-induced lysis by natural killer cells

We investigated the lysis of fresh human solid tumor cells by peripheral blood T lymphocytes in the presence of lectins and anti-CD3 monoclonal antibodies (mAb). Addition of certain lectins (Con A, PHA, or WGA) directly into the 4-hr 51Cr-release assay caused significant lysis of (P less than 0.001) noncultured solid tumor targets by enriched populations of granular lymphocytes (GL). Significant levels (P at least less than 0.001) of Con A- or PHA-dependent solid tumor lysis by GL-enriched lymphocytes were observed in 32 of 39 donors (82%) and 14 of 20 donors (70%), respectively. In contrast, the addition of other lectins (PNA, PWM, or LPS) or anti-CD3 mAb did not cause cytotoxicity. The levels of Con A-dependent lysis were comparable to those of interleukin 2 (IL-2)-induced lysis by Leu 11b+ natural killer (NK) cells. The presence of lectins at the effector phase, but not of recombinant IL-2 (rIL-2), was required for the lysis of solid tumor targets. Both Con A-dependent and rIL-2-induced lysis were totally inhibited by treatment of the effector cells with the lysosomotropic agent L-leucine methyl ester (LeuOMe). Effector cells responsible for Con A-dependent lysis of solid tumors expressed T3 (CD3), T8 (CD8), and Leu 7 antigens, but lacked T4 (CD4) and Leu 11 (CD16) antigens as determined by both negative and positive cell selection studies. Con A-dependent lysis was inhibited at the effector phase by anti-CD3 (OKT3 or anti-Leu 4) or anti-CD2 (OKT11) mAb. On the basis of their phenotype (Leu 7+ CD3+ CD8+ CD16-), we hypothesize that these effector cells may contain a population of cytotoxic T cells (CTL) generated in vivo against autologous modified cells that can lyse fresh solid tumor target cells under conditions where the recognition requirements for the CTL are bypassed by lectin approximation.

Characterization of a Subset of Human Natural Killer Cells That Express OKM1 But Lack HNK-1 (Leu-7) Antigens

The HNK‐1(Leu‐7) monoclonal antibody selectively identifies a population of human granular lymphocytes with natural killer (NK) cell activity. We previously reported that the HNK‐l+ cell fraction purified from blood mononuclear cells accounted for virtually all NK activity in six individuals. In this study we analysed additional normal individuals and found that in eight out of 14 donors HNK‐1+ cells, purified with a fluorescence‐activated cell sorter (FACS). exhibited greatly enriched NK cell activity, whereas HNK‐1 cells did not have significant activity. In four donors the HNK‐1+ cells were enriched in NK activity compared with HNK‐1‐ cells; however, the HNK‐1‐ cells also had moderate levels of activity. In the two remaining donors. NK activity was not enriched in the HNK‐1+ fraction in comparison with the HNK‐1‐ fraction. To determine the cell type responsible for NK activity in the HNK‐1 subset, these cells were further sorted with the FACS into OKM1+ and OMK1‐ tractions and analysed for morphology and function. HNK‐1‐ OKM1‐ cells were found to he small‐ to medium‐sized lymphocytes devoid of NK activity in all donors tested, whereas most HNK‐1‐ OKM1‐ cells were granular lymphocytes and in some donors demonstrated NK function at a level comparable to HNK‐1+ cells. Thus some individuals have an important subset of granular lymphocytes with NK‐cell activity and the HNK‐1‐ OKMl+ phenotype. It is important to account for these cells in studies involving granular lymphocytes and NK cell function.