Immunotherapy of established tumors in mice by intratumoral injection of interleukin-2 plasmid DNA: induction of CD8+ T-cell immunity.

Intratumoral (i.t.) injection of a plasmid DNA vector encoding the murine interleukin-2 (IL-2) gene was used to treat established renal cell carcinoma (Renca) tumors in BALB/c mice. Tumor regression was observed in 60-90% of mice that were injected i.t. for 4 days with IL-2 plasmid DNA complexed with the cationic lipid DMRIE/DOPE ((+/-)-N-(2-hydroxyethyl)-N,N-dimethyl-2,3-bis(tetradecyloxy)-1-propa naminium bromide/dioleoylphosphatidylethanolamine). The mice remained tumor-free until the conclusion of the study, which was 4 months after tumor challenge. In a rechallenge experiment, mice that were rendered tumor-free for 6 months by IL-2 plasmid DNA treatment rejected a subsequent challenge of Renca cells but could not reject a challenge with the unrelated, syngeneic CT-26 tumor. Spleen cells from cured mice contained Renca-specific cytotoxic T lymphocytes, and adoptive transfer of mixed lymphocyte cultures into naive mice at 2 days after challenge with Renca cells prevented tumor growth. In vivo depletion of T-cell subsets at the time of i.t. injection with IL-2 plasmid DNA demonstrated that CD8+ T cells, but not CD4+ T cells, were the primary effectors of the antitumor response.

Immunization with plasmid DNA using a pneumatic gun.

We characterize a method by which the Med-E-Jet pneumatic vaccination gun can be used to propel intact, supercoiled plasmid DNA through skin and into skeletal muscles of mice. Intramuscular injection of plasmids containing the firefly luciferase gene linked to the human cytomegalovirus promoter resulted in the expression of several hundred picograms of luciferase enzyme in quadriceps muscles. Intramuscular injections of a plasmid containing the influenza A nuclear protein gene regulated by the same promoter resulted in the generation of potent and specific anti-nuclear protein humoral and cellular immune responses. This convenient and rapid injection method would be well-suited for genetic immunization of humans.

Characterization of a potential marker of corneal epithelial stem cells.

Corneal epithelial stem cells are thought to be localized in the basal cell layer of the limbus. We developed a monoclonal antibody designated 4G10.3 that immunolocalized to limbal basal cells in rat corneas. Western blot analysis demonstrated that 4G10.3 reacted with a single band of 50,000 molecular weight in rat and rabbit corneal epithelium and 48,000-49,000 in human epithelium. Following extraction of corneal epithelium in 20 mM Tris-HCl (pH 6.8) and ultracentrifugation at 100,000 x g, the 50-kD protein was detected in the soluble fraction. 4G10.3 also was used to examine the response of the limbal basal cells to epithelial debridement and thermal burn wounds in the rat. In unwounded control corneas, 40.8 +/- 12.0 (mean +/- standard deviation) cell per limbal area bound 4G10.3. Following a 3 mm debridement wound, the number of cells binding 4G10.3 increased to 77.0 +/- 16.9 two days post-injury and returned to control levels by three days. Following thermal burn, 36.2 +/- 11.2, 68.8 +/- 15.8, 85.4 +/- 15.4, 104.6 +/- 13.8, and 88.0 +/- 40.2 cells per limbal area bound 4G10.3 18 hours, and 1, 2, 3, and 7 days post-injury, respectively. The 50 kD protein and 4G10.3 antibody provided a biochemical and immunological marker of limbal basal cells, hypothesized to be corneal epithelial stem cells.

Long-term anti-nucleoprotein cellular and humoral immunity is induced by intramuscular injection of plasmid DNA containing NP gene.

Cytolytic T-lymphocyte-mediated killing is thought to be an important effector mechanism in controlling viral infections. Recently, we reported that intramuscular injection of plasmid DNA containing the nucleoprotein (NP) gene of the influenza virus resulted in generating nucleoprotein-specific cytolytic T cells and antibodies. Gene-injected mice were subsequently protected from a lethal challenge with live influenza virus. Here we show that a single intramuscular injection of a small dose of nucleoprotein plasmid DNA generates nucleoprotein-specific cellular and humoral immune responses that last 1 year. The cellular response is associated with the CD8+ subpopulation of T cells. Thus, plasmid DNA injections can be used to induce long-lasting immune responses against the viral gene product without an exposure to live virus itself.

Characterization of retinal pigment epithelial cells cultured on microporous filters.

Retinal pigment epithelium (RPE) cultured on microporous filter supports was compared to RPE cultured on plastic and evaluated for features characteristic of RPE in vivo. RPE cells grown on filters were cuboidal, formed junctional complex structures between cells, and had elaborate microvilli and basal infoldings similar to RPE in vivo, while RPE grown on plastic also formed intercellular junctions but appeared squamous and had few microvilli and basal infoldings. RPE grown on filters or plastic secreted an extracellular matrix at the basal surface and ingested isolated rat rod outer segments at the apical surface. RPE grown on filters coated with laminin or fibronectin became confluent more rapidly than RPE grown on uncoated filters, while RPE grown at the same density on filters coated with collagen type I did not become confluent. The laminin and fibronectin coatings did not alter the RPE cell morphology; however, cells seeded on collagen-coated filters grew in large disorganized clusters. RPE grown on laminin-coated filters formed functional tight junctions as evidenced by the capacity of RPE monolayers to prevent the bulk flow of medium and the passage of trypan blue across the filter. Radiolabeled sucrose and inulin were used to measure the paracellular flux through the tight junctions between cells. The passage of these tracers was linear over time, with the lower molecular weight tracer, sucrose, passing through the monolayer more readily than inulin. Values for the flux of radiolabeled bovine serum albumin across RPE monolayers fell between values for sucrose and inulin. The results from these studies show that RPE monolayers cultured on laminin-coated filters maintain a morphology similar to that of RPE in vivo, are capable of ingesting rod outer segments, and form a selectively permeable barrier to various tracers. This culture system should be useful for studies of transepithelial transport, secretion, endocytosis and exocytosis that require independent control of the extracellular environment at the apical and basolateral cell surfaces.

Alpha-enolase is restricted to basal cells of stratified squamous epithelium.

We have developed a monoclonal antibody against a 50-kDa protein that binds preferentially to basal cells in the limbus of rat, rabbit, and human corneas (J. D. Zieske, G. Bukusoglu, and M. A. Yankauckas, Invest. Ophthalmol. Visual Sci. 33, 143-152, 1992). Here we report on the purification and identification of the antigen. The 50-kDa antigen was purified from rabbit limbal and corneal epithelium using HPLC methodology including anion exchange (DEAE) followed by reverse-phase (C18) chromatography. The purified 50-kDa protein was then digested with endoproteinase Lys-C, and a reproducible profile comprising approximately 20 peptides was observed by reverse-phase HPLC of the digest. Sequence analysis of five peptides ranging in length from 4 to 20 residues revealed that the 50-kDa protein was alpha-enolase, a glycolytic enzyme. Overall, 57 amino acids were identified with a 95% sequence homology. Localization of alpha-enolase in rat epithelium by immunofluorescence microscopy demonstrated that simple epithelium contained low or undetectable levels of the enzyme. Stratified squamous epithelium, however, showed high levels of alpha-enolase, which was localized specifically to cells of the basal layer. Epidermal, corneal limbal, oral mucosal, vaginal, and laryngeal epithelium all showed cytoplasmic binding specific to the basal cells. These data indicate that the glycolytic enzyme alpha-enolase is preferentially localized in the basal cell layer of stratified squamous epithelium and suggest that glycolytic activity is concentrated in these cells. The localization pattern suggests that a major change in metabolism occurs as cells leave the mitotically active basal cell layer and migrate toward terminal differentiation in the suprabasal cell layers.

Intradermal gene immunization: the possible role of DNA uptake in the induction of cellular immunity to viruses.

The skin and mucous membranes are the anatomical sites were most viruses are first encountered by the immune system. Previous experiments have suggested that striated muscle cells are unique among mammalian cell types in their capacity to take up and express free DNA in the absence of a viral vector or physical carrier. However, we have found that mice injected into the superficial skin with free (naked) plasmid DNA encoding the influenza nucleoprotein gene had discrete foci of epidermal and dermal cells, including cells with dendritic morphology, that contained immunoreactive nucleoprotein antigen. A single intradermal administration of 0.3-15 micrograms of free plasmid DNA induced anti-nucleoprotein-specific antibody and cytotoxic T lymphocytes that persisted for at least 68-70 weeks after vaccination. Intradermal gene administration induced higher antibody titers than did direct gene injection into skeletal muscle and did not cause local inflammation or necrosis. Compared with control animals, the gene-injected mice were resistant to challenge with a heterologous strain of influenza virus. These results indicate that the cells of the skin can take up and express free foreign DNA and induce cellular and humoral immune responses against the encoded protein. We suggest that DNA uptake by the skin-associated lymphoid tissues may play a role in the induction of cytotoxic T cells against viruses and other intracellular pathogens.