In vivo activity of tumor necrosis factor (TNF) mutants. Secretory but not membrane-bound TNF mediates the regression of retrovirally transduced murine tumor.

We have previously demonstrated that murine tumor cells transduced with a retrovirus containing the cDNA encoding wild-type human TNF regress in vivo when injected into immunocompetent mice; this regression is T cell mediated. To determine whether membrane-associated or secreted TNF was responsible for tumor regression, we transduced a cloned murine fibrosarcoma 205 F4 with retroviruses encoding modified human TNF genes. The cloned tumor lines of one retroviral transduction expressed only membrane bound 26-kDa TNF. This TNF could not be cleaved or secreted, but was present on the cell surface. A second retrovirus caused the expression of only secretory 17-kDa TNF, as the transmembrane domain of the cDNA was deleted. The TNF produced by tumor cells transduced with either retroviral vector was functional in vitro as direct lysis of the TNF-sensitive target L929 by transduced tumor cells was demonstrated. The TNF present on 26-kDa expressing tumors was membrane bound as supernatants from cultured 17-kDa TNF expressing tumor cells but not 26-kDa TNF expressing tumors mediated the lysis of L929 cells. Both tumors were injected s.c. into syngeneic mice and tumor growth was measured serially. In repeated experiments, 26-kDa TNF expressing tumors grew progressively in all mice. In contrast, 17-kDa TNF expressing tumors grew for 10 days and then regressed with all animals free of tumor at 28 days. Tumor regression was abrogated by in vivo injection of an anti-TNF antibody. Similar results were obtained in a second tumor model, 203 E4. Thus regression of TNF transduced tumors in vivo requires secretion of TNF, as membrane-bound TNF is insufficient to elicit the host response.     

Impact of cytokine administration on the generation of antitumor reactivity in patients with metastatic melanoma receiving a peptide vaccine.

Patients with metastatic melanoma were immunized with an immunodominant peptide derived from the gp100 melanoma-melanocyte differentiation Ag that was modified to increase binding to HLA-A+0201. A total of 10 of 11 patients who received the g209-2M peptide alone developed precursors reactive with the native g209 peptide, compared with only 5 of 16 patients who received g209-2M peptide plus IL-2 (p2 = 0.005). Peptide reactivity closely correlated with the recognition of HLA-A+0201 melanoma cells (p < 0. 001). The decrease in immune reactivity when peptide was administered with IL-2 appeared specific for the immunizing peptide, since reactivity to an influenza peptide resulting from prior exposure was not affected. Preexisting antitumor precursors did not decrease when peptide plus IL-2 was administered. The administration of GM-CSF or IL-12 also resulted in a decrease in circulating precursors compared with the administration of peptide alone, though not as great a decrease as that seen with IL-2. Immunization with peptide plus IL-2 did, however, appear to have clinical impact since 6 of the 16 patients (38%) that received peptide plus IL-2 had objective cancer regressions. It thus appeared possible that immunization with peptide plus IL-2 resulted in sequestering or apoptotic destruction of newly activated immune cells at the tumor site. These represent the first detailed studies of the impact of immunization with tumor peptides in conjunction with a variety of cytokines in patients with metastatic cancer.     

Alphavirus-based DNA vaccine breaks immunological tolerance by activating innate antiviral pathways

Cancer vaccines targeting 'self' antigens that are expressed at consistently high levels by tumor cells are potentially useful in immunotherapy, but immunological tolerance may block their function. Here, we describe a novel, naked DNA vaccine encoding an alphavirus replicon (self-replicating mRNA) and the self/tumor antigen tyrosinase-related protein-1. Unlike conventional DNA vaccines, this vaccine can break tolerance and provide immunity to melanoma. The vaccine mediates production of double-stranded RNA, as evidenced by the autophosphorylation of dsRNA-dependent protein kinase R (PKR). Double-stranded RNA is critical to vaccine function because both the immunogenicity and the anti-tumor activity of the vaccine are blocked in mice deficient for the RNase L enzyme, a key component of the 2',5'-linked oligoadenylate synthetase antiviral pathway involved in double-stranded RNA recognition. This study shows for the first time that alphaviral replicon-encoding DNA vaccines activate innate immune pathways known to drive antiviral immune responses, and points the way to strategies for improving the efficacy of immunization with naked DNA.     

Cancer therapy using a self-replicating RNA vaccine.

'Naked' nucleic acid vaccines are potentially useful candidates for the treatment of patients with cancer, but their clinical efficacy has yet to be demonstrated. We sought to enhance the immunogenicity of a nucleic acid vaccine by making it 'self-replicating'. We accomplished this by using a gene encoding an RNA replicase polyprotein derived from the Semliki forest virus, in combination with a model antigen. A single intramuscular injection of a self-replicating RNA immunogen elicited antigen-specific antibody and CD8+ T-cell responses at doses as low as 0.1 microg. Pre-immunization with a self-replicating RNA vector protected mice from tumor challenge, and therapeutic immunization prolonged the survival of mice with established tumors. The self-replicating RNA vectors did not mediate the production of substantially more model antigen than a conventional DNA vaccine did in vitro. However, the enhanced efficacy in vivo correlated with a caspase-dependent apoptotic death in transfected cells. This death facilitated the uptake of apoptotic cells by dendritic cells, providing a potential mechanism for enhanced immunogenicity. Naked, non-infectious, self-replicating RNA may be an excellent candidate for the development of new cancer vaccines.     

Partial replication of UV-irradiated T4 bacteriophage DNA results in amplification of specific genetic areas.

Upon infection of Escherichia coli with bromodeoxyuridine-labeled t4 phage that had received 10 lethal hits of UV irradiation, a sizable amount of phage DNA was synthesized (approximately 36 phage equivalent units of DNA per infected bacterium), although very little multiplicity reactivation occurs. This progeny DNA was isolated and analyzed. This DNA was biased in its genetic representation, as shown by hybridization to cloned segments of the T4 genome immobilized on nitrocellulose filters. Preferentially amplified areas corresponded to regions containing origins of T4 DNA replication. The size of the progeny DNA increased with time after infection, possibly due to recombination between partial replicas and nonreplicated subunits or due to the gradual overcoming of the UV damage. As the size of the progeny DNA increased, all of the genes were more equally represented, resulting in a decrease in the genetic bias. Amplification of specific genetic areas was also observed upon infection with UV-irradiated, nonbromodeoxyuridine-substituted (light) phage. However, the genetic bias observed in this case was not as great as that observed with bromodeoxyuridine-substituted phage. This is most likely due to the higher efficiency of multiplicity reactivation of the light phage.     

Cutting edge: CD4+ T cell control of CD8+ T cell reactivity to a model tumor antigen

Neoantigens resulting from the inherent genomic instability of tumor cells generally do not trigger immune recognition. Similarly, transfection of tumors with model Ags often fails to elicit CD8+ T cell responses or alter a tumor's growth rate or lethality. We report here that the adoptive transfer of activated Th1-type CD4+ T cells specific for a model tumor Ag results in the de novo generation of CD8+ T cells with specificity to that Ag and concomitant tumor destruction. The anti-tumor effects of the CD4+ T cells required the presence of both MHC class I and class II on host cells, as evidenced by experiments in knockout mice, suggesting that CD4+ T cells enhanced the ability of host APC to activate endogenous CD8+ T cells. These results indicate that the apparent inability of tumor cells expressing highly immunogenic epitopes to activate tumor-specific CD8+ T cells can be altered by activated CD4+ T cells.     

A new practical tool for deriving a functional signature for herbaceous vegetation

Hypothesis: For any one time and place a ‘functional signature’ can be derived for a sample of herbaceous vegetation in a way that concisely represents the balance between the different clusters of functional attributes that are present among component species. Methods: We developed a spreadsheet‐based tool for calculating functional signatures within the context of the C‐S‐R system of plant functional types. We used the tool to calculate and compare signatures for specimen British vegetation samples which differed in management regime and location in time. Conclusion: The integrative power of the ‘C‐S‐R signature’ is useful in comparative studies involving widely differing samples. Movements in the signature can be used to indicate degree of resistance, resilience, eutrophication and dereliction. Systems of plant functional types other than C‐S‐R might also be approached in this way. Availability: The tool can be downloaded free of charge from the first author's web pages or from the journal's electronic archive.     

Mutations in a steroid hormone-regulated gene disrupt the metamorphosis of the central nervous system in Drosophila

The actions of steroid hormones on vertebrate and invertebrate nervous systems include alterations in neuronal architecture, regulation of neuronal differentiation, and programmed cell death. In particular, central nervous system (CNS) metamorphosis in insects requires a precise pattern of exposure to the steroid molting hormone 20-hydroxyecdysone (ecdysterone). To test whether the effects of steroid hormones on the insect nervous system are due to changes in patterns of gene expression, we examined Drosophila mutants of the ecdysterone-regulated locus, the Broad Complex (BR-C). This report documents aspects of CNS reorganization which are dependent on BR-C function. During wild-type metamorphosis, CNS components undergo dramatic morphogenetic movements relative to each other and to the body wall. These movements, in particular, the separation of the subesophageal ganglion from the thoracic ganglion, the positioning of the developing visual system, and the fusion of right and left brain hemispheres, are deranged in BR-C mutants. In addition, a subset of mutants shows disorganization of optic lobe neuropil, both within and among optic lobe ganglia. Optic lobe disorganization is found in mutants of the br and l(1)2Bc complementation groups, but not in those of the rbp complementation group. This suggests that the three complementation groups of this complex locus represent distinct but overlapping functions necessary for normal CNS reorganization. This study demonstrates that ecdysterone-regulated gene expression is essential for CNS metamorphosis, illustrating the utility of Drosophila as a model system for investigating the genetic basis of steroid hormone action on the nervous system.     

Unopposed production of granulocyte-macrophage colony-stimulating factor by tumors inhibits CD8+ T cell responses by dysregulating antigen-presenting cell maturation

Tumor cells gene-modified to produce GM-CSF potently stimulate antitumor immune responses, in part, by causing the growth and differentiation of dendritic cells (DC). However, GM-CSF-modified tumor cells must be gamma-irradiated or they will grow progressively, killing the host. We observed that 23 of 75 (31%) human tumor lines and two commonly used mouse tumor lines spontaneously produced GM-CSF. In mice, chronic GM-CSF production by tumors suppressed Ag-specific CD8+ T cell responses. Interestingly, an inhibitory population of adherent CD11b(Mac-1)/Gr-1 double-positive cells caused the observed impairment of CD8+ T cell function upon direct cell-to-cell contact. The inhibitory cells were positive for some markers associated with Ag presenting cells, like F4/80, but were negative for markers associated with fully mature DC like DEC205, B7. 2, and MHC class II. We have previously reported that a similar or identical population of inhibitory "immature" APC was elicited after immunization with powerful recombinant immunogens. We show here that these inhibitory cells can be elicited by the administration of recombinant GM-CSF alone, and, furthermore, that they can be differentiated ex vivo into "mature" APC by the addition of IL-4 and GM-CSF. Thus, tumors may be able to escape from immune detection by producing "unopposed" GM-CSF, thereby disrupting the balance of cytokines needed for the maturation of fully functional DC. Further, CD11b/Gr-1 double-positive cells may function as "inhibitory" APC under the influence of GM-CSF alone.