Antitumoral effect of a nonviral interleukin-2 gene therapy is enhanced by combination with 5-fluorouracil.

Using a novel cationic lipid delivery system consisting of N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride and cholesterol, we delivered murine interleukin-2 (IL-2) cDNA directly into an established murine renal cell carcinoma (Renca). Production of IL-2 within the tumor induced rejection of established tumors (62% on average), whereas control plasmid had little or no effect (17% on average). Surviving animals treated with IL-2:lipid were highly resistant to Renca rechallenge, but not to cross-challenge with a syngeneic mammary adenocarcinoma. Experiments on selectively immunosuppressed animals indicated a requirement for CD8+ T, natural killer, and polymorphonuclear cells. By contrast, depletion of CD4+ T cells did not disrupt the ability of IL-2:lipid to induce tumor rejection. A combination of IL-2 gene therapy with 5-fluorouracil treatment increased the antitumoral efficacy and survival of mice bearing primary and metastatic Renca tumors (42% survival with IL-2:lipid compared with 94% survival with IL-2:lipid plus 5-fluorouracil). These data indicate that rejection of primary and metastatic tumors can be achieved after intratumoral delivery of a nonviral IL-2 gene therapy, and is increased in combination with systemic delivery of a conventional chemotherapeutic agent.

Detachment and lysis of adherent target cells by CD4+ T cell clones involve multiple effector mechanisms.

Detachment of adherent targets from their substratum is a unique activity of both CD8+ and CD4+ T cells. Recently, we have demonstrated that with mixed, alloreactive populations, CD4+ T-cell-mediated detachment is kinetically distinct from lysis and requires protein synthesis. Heterogeneity of effector phenotypes precluded further elucidation of the mechanism of detachment at the mixed population level. Here, we examine further the mechanism of target cell detachment by CD4+ cells using clones which differ in lytic efficiency and demonstrate that detachment of adherent targets from their substratum (1) may result from either protein synthesis-dependent or independent pathways, which can be correlated with differences in clonal lytic phenotype, (2) is contact dependent and does not involve soluble factors, (3) can be pharmacologically uncoupled from IL4 production, as a measure of cytokine release, and (4) fails to correlate with perforin expression by immunocytochemistry. Finally, isolated granule preparations are unable to mediate detachment independent from lysis.

Molecular characterization of the interleukin-8 receptor.

Recently a rabbit cDNA (F3R) was characterized as binding and causing calcium mobilization induced by the formyl-methionine-leucine-phenylalanine peptide (fMLP). In the study reported here, cloned DNAs were isolated from rabbit genomic DNA by PCR based on the sequence of F3R. The cloned DNAs have several differences in the DNA sequence compared to the reported F3R sequence that alter the predicted protein sequence. COS-7 cells transfected with these clones in a mammalian expression vector bind human IL-8 with high affinity, but do not bind fMLP. We therefore believe that the cDNAs isolated encode the rabbit IL-8 receptor.

Direct evidence for an intracellular role for tumor necrosis factor-alpha 1. Microinjection of tumor necrosis factor kills target cells.

TNF-alpha is a small peptide cytokine produced primarily by activated macrophages. One of the many biologic activities of TNF is the killing of diverse types of tumor cells. We considered the possibility that killing was mediated by TNF itself at an intracellular site, subsequent to receptor-mediated endocytosis. To test this hypothesis, we microinjected TNF into various murine normal cells and cell lines, some of which were killed by TNF given by the usual extracellular route, and others that were not. Cytotoxic effects of microinjected TNF were observed in several cell types 2 to 4 h after injection. L929 fibroblasts were killed by either extracellular or intracellular TNF. A TNF-resistant subline of L929 was insensitive to either extracellular or intracellular TNF. L6 fibroblasts were found to be resistant to high doses of TNF given either extracellularly or microinjected. Normal macrophages and the J774 macrophage-like cell line were not killed by extracellular TNF, but were rapidly killed by microinjected TNF. Thus, TNF, an extracellular peptide ligand, has an intracellular activity, suggesting that internalization of this ligand may have important intracellular biochemical roles.

The toxicity of rat large granular lymphocyte tumor cells and their cytoplasmic granules for rodent and African trypanosomes.

To explain previous findings that rodent and African trypanosomes are relatively insusceptible to the actions of NK cells, their sensitivity to the cytotoxicity of rat LGL tumor cells and isolated cytolysin-containing granules was studied. LGL tumor cells displayed modest spontaneous killing of rodent trypanosomes but were considerably more effective in the antibody-dependent cell-mediated cytotoxicity mode in the presence of specific antibody. The trypanosomes were quite resistant to lysis by the cytolysin-containing granules, compared with other types of cells. The slow inefficient lysis that occurred in the presence of divalent cations involved granule concentrations thousands of times greater than was required for lysis of SRBC. Rodent trypanosomes were significantly more susceptible to lysis when divested of their surface glycoprotein coats. In the absence (or near absence) of divalent cations, a substance (or substances) present in the granules rapidly destroyed intact and nude trypanosomes; this activity probably was not associated with cytolysin. The rate and efficiency with which the divalent cation-independent substance destroyed three species of trypanosomes indicate that this material deserves further study with an eye to its potential use as a therapeutic agent.

Possible involvement of CTL granule proteases in target cell DNA breakdown.

We have carried out experiments to test whether the granule exocytosis model for lymphocyte cytotoxicity can account for the rapid target DNA breakdown seen during CTL-induced cytotoxicity. Dense granules isolated from cloned mouse CTL and from rat NK tumor cells cause target DNA breakdown during granule cytolysin-mediated lysis of tumor cells, while the purified granule cytolysin caused lysis without DNA breakdown. When target cells are permeabilized with detergent, granule extracts have the ability to release 125I-DNA from nuclei in the absence of detectable cytolysin activity. This activity formed the basis for a nuclear DNA release (NDR) assay; this activity was a property of dense granules of cytotoxic lymphocytes but generally not of other types of lymphoid cells. NDR activity in NK tumor granules had a pH optimum of 7 and was inhibited by micromolar levels of Zn+2, and could be purified away from the granule cytolysin by column chromatography. NDR activity in CTL dense granules could be inactivated by submillimolar concentrations of the protease inhibitors PMSF and DFP (but not soybean trypsin inhibitor or TLCK). In support of the relevance to CTL cytotoxicity of these findings with the NDR assay, pretreatment of CTL with PMSF in the presence of agents raising the intragranular pH inactivated 125I-DNA release from target cells (but not the 51Cr release). These results suggest that a CTL granule component(s), probably a protease, is required for target DNA breakdown.

Biochemical and functional properties of serine esterases in acidic cytoplasmic granules of cytotoxic T lymphocytes.

Percoll gradient fractions of homogenates of murine cloned cytotoxic T lymphocytes (CTL) were analyzed for the trypsin-like enzyme alpha-N-benzyloxy-carbonyl-L-lysinethiobenzyl ester (BLT) esterase recently described in CTL homogenates. Enzymatic activity was found in three areas of the gradient: the dense cytolysin containing granules; a light granule fraction; and a variable amount in the soluble fraction at the top of the gradient. Gel filtration columns showed a major peak of BLT esterase activity eluted at the position of a 60-kDa protein, and an additional, minor BLT esterase peak eluting at about 27 kDa. The separated enzymes were both significantly inhibited by the serine protease inhibitors diisopropylfluorophosphate and phenylmethyl sulfonyl fluoride (PMSF), indicating they are both serine proteases, but showed different patterns of inhibition by a series of inhibitors, suggesting the larger enzyme is not a simple dimer of the smaller. pH activity profiles of both CTL BLT esterases showed an optimum at about pH 8. PMSF inactivation of BLT esterase in detergent extracts of CTL diminished sharply as the pH was dropped below 7. Agents which raise the pH of acidic intracellular compartments were found to markedly enhance the PMSF inactivation of BLT esterase in intact CTL, showing that the granules have a low internal pH. Similarly, [3H]diisopropylfluorophosphate labeling of intact CTL gave four protein bands on non-reduced gels, of which two were labeled threefold more effectively in the presence of chloroquine. In parallel studies of inactivation of CTL lytic activity, PMSF pretreatment caused a 50% reduction of the lytic activity under conditions where greater than 90% of the BLT esterase activity was inactivated. Addition of agents raising the intragranular pH dramatically enhanced the BLT esterase inactivation but did not concomitantly reduce CTL lytic activity. These results indicate that inactivation of lytic function by PMSF is unlikely to be due to its reaction with protease in acidic granules, and suggest that the activity of these enzymes may not be required for cytotoxicity.

Effect of phorbol esters on alloimmune cytolysis.

The tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) profoundly affects cytolytic T-lymphocyte activity. Alloimmune C57BL/6 (H-2b anti-H-2d) cytolytic splenocytes treated with TPA, 0.3 to 3.0 ng/ml, killed specific P815 (H-2d) targets significantly better than did untreated controls as measured in 4-hr 51Cr release microcytotoxicity assay. Higher concentrations of TPA in the 30- to 100-ng/ml range significantly inhibited cytolytic function. The non-tumor-promoting analog, 4 alpha-phorbol-12,13-didecanoate, failed to affect killing at all doses tested. TPA-induced augmentation of cytolytic function requires an immunologically sensitized splenocyte population, since normal nonimmunized splenocytes treated with TPA did not kill target cells. Furthermore, treatment of splenocytes with anti-Thy 1,2 antibody and complement abrogated killing, indicating that T-lymphocytes mediate the killing. The TPA-induced effect does not require macrophage-like cells, since augmented killing occurred despite the removal of glass-adherent or iron-phagocytosing cells. Finally, the cytolytically augmented effector cells remain immunologically specific, since the nonspecific targets, syngeneic EL4 (H-2b) and third-party L929 (H-2k), are not killed. Thus, low levels of TPA augment the cytolytic ability of alloimmune T-lymphocytes against their specific target cells.