Immortalization of human corneal epithelial cells using simian virus 40 large T antigen and cell characterization.

INTRODUCTION: Primary cultures of human corneal epithelial (HCE) cells usually cease to grow after four or five passages. This result in a small cell yield for experiments such as the eye irritancy test represents a serious problem for human and animal corneal epithelial research. In the present study, we established an HCE cell line immortalized by simian virus 40 (SV40), a polyomavirus, and characterized the inherent morphologic and cytologic cell properties. METHODS: Primary cultured HCE cells were infected with a SV40 large T antigen (SV40 T)-expressing retrovirus, and were selected using G418 solution, an aminoglycoside antibiotic. To ensure that the immortalized cell lines express SV40 T and cytokeratin-3, a corneal epithelial-specific marker, we conducted reverse-transcription (RT)-PCR and Western blot analysis. RESULTS: These cell lines continued to grow for more than 50 generations, exhibiting a cobble stone-like appearance similar to normal HCE cells and an increased proliferation rate compared to primary cultured HCE cells. RT-PCR results showed that the immortalized cell lines expressed SV40 T while the primary cultured cells did not. In the Western blot assay, protein levels of phosphorylated (Ser15) p53 protein were significantly decreased in the immortalized cell lines while the expression of total p53 protein was constant. In addition, expression of p21(cip1), a cell cycle protein, was down-regulated in the immortalized cells. Moreover, a cornea epithelium-specific marker, cytokeratin-3 (CK-3), was expressed at equal levels in the immortalized cells and primary HCE cells. DISCUSSION: Taken together, these results indicate that immortalized HCE cell lines were successfully established using the SV40-retroviral vector. These cells may be an excellent model for detecting the adverse effects of standard toxic materials and could replace the traditional eye irritation test as an animal-free alternative method.

Effects of route of inoculation and viral genetic variation on antibody responses to polyomavirus SV40 in Syrian golden hamsters.

Genetic variants of polyomavirus SV40 are powerful agents with which to define viral effects on cells and carcinogenesis pathways. We hypothesized that differences in biologic variation among viral strains affect the process of viral infection and are reflected in antibody responses to the viral nonstructural large T-antigen (TAg) protein but not in neutralizing antibody responses against the inoculated viral particles. We analyzed the production of TAg antibody and neutralizing antibody in Syrian golden hamsters that were inoculated with SV40 viral strains by intracardiac, intravenous, or intraperitoneal routes and remained tumor free. Compared with the intraperitoneal route, intravascular (that is, intravenous, intracardiac) inoculation resulted in increased frequency of responsiveness to TAg but not in higher TAg antibody titers. The intravascular route was superior both for eliciting neutralizing antibody responses and for higher titers of those responses. Viruses with complex regulatory regions induced TAg antibody more often than did viruses with simple regulatory regions after intraperitoneal but not intravascular injections, with no differences in antibody titers. This viral genetic variation had no effect on neutralizing antibody production after intraperitoneal or intravascular inoculations or on neutralizing antibody titers achieved. These findings confirm that SV40 variants differ in their biologic properties. Route of inoculation combined with viral genetic variation significantly influence the development of serum antibodies to SV40 TAg in tumor-free hamsters. Route of inoculation-but not viral genetic variation-is an important factor in production of neutralizing antibody to SV40.

Inefficient cross-presentation limits the CD8+ T cell response to a subdominant tumor antigen epitope.

CD8(+) T lymphocytes (T(CD8)) responding to subdominant epitopes provide alternate targets for the immunotherapy of cancer, particularly when self-tolerance limits the response to immunodominant epitopes. However, the mechanisms that promote T(CD8) subdominance to tumor Ags remain obscure. We investigated the basis for the lack of priming against a subdominant tumor epitope following immunization of C57BL/6 (B6) mice with SV40 large tumor Ag (T Ag)-transformed cells. Immunization of B6 mice with wild-type T Ag-transformed cells primes T(CD8) specific for three immunodominant T Ag epitopes (epitopes I, II/III, and IV) but fails to induce T(CD8) specific for the subdominant T Ag epitope V. Using adoptively transferred T(CD8) from epitope V-specific TCR transgenic mice and immunization with T Ag-transformed cells, we demonstrate that the subdominant epitope V is weakly cross-presented relative to immunodominant epitopes derived from the same protein Ag. Priming of naive epitope V-specific TCR transgenic T(CD8) in B6 mice required cross-presentation by host APC. However, robust expansion of these T(CD8) required additional direct presentation of the subdominant epitope by T Ag-transformed cells and was only significant following immunization with T Ag-expressing cells lacking the immunodominant epitopes. These results indicate that limited cross-presentation coupled with competition by immunodominant epitope-specific T(CD8) contributes to the subdominant nature of a tumor-specific epitope. This finding has implications for vaccination strategies targeting T(CD8) responses to cancer.

Autoimmunity to DNA and nucleosomes in binary tetracycline-regulated polyomavirus T-Ag transgenic mice.

The mechanism(s) responsible for autoimmunity to DNA and nucleosomes in SLE is largely unknown. We have demonstrated that nucleosome-polyomavirus T-Ag complexes, formed in context of productive polyomavirus infection, activate dsDNA-specific B cells and nucleosome-specific CD4(+) T cells. To investigate whether de novo expressed T-Ag is able to terminate nucleosome-specific T cell tolerance and to maintain anti-dsDNA Ab production in nonautoimmune mice, we developed two binary transgenic mouse variants in which expression of SV40 large T-Ag is controlled by tetracycline, MUP tTA/T-Ag (tet-off), and CMV rtTA/T-Ag (tet-on) mice. Data demonstrate that MUP tTA/T-Ag mice, but not CMV rtTA/T-Ag mice, are tightly controlling T-Ag expression. In MUP tTA/T-Ag transgenic mice, postnatal T-Ag expression activated CD8(+) T cells but not DNA-specific B cells, while immunization with T-Ag and nucleosome-T-Ag-complexes before T-Ag expression resulted in elevated and remarkably stable titers of anti-T-Ag and anti-dsDNA Abs and activation of T-Ag-specific CD4(+) T cells. Immunization of nonexpressing MUP tTA/T-Ag mice resulted in transient anti-T-Ag and anti-dsDNA Abs. This system reveals that a de novo expressed DNA-binding quasi-autoantigen maintain anti-dsDNA Abs and CD4(+) T cell activation once initiated by immunization, demonstrating direct impact of a single in vivo expressed molecule on sustained autoimmunity to DNA and nucleosomes.

Effective genetic vaccination with a widely shared endogenous retroviral tumor antigen requires CD40 stimulation during tumor rejection phase.

Endogenous retrovirus (ERV) products are recognized by T lymphocytes in mice and humans. As these Ags are preferentially expressed by neoplastic tissues, they might represent an ideal target for active immunization by genetic vaccination. However, i.m. inoculation of plasmid DNA encoding mouse gp70 or p15E, two products of the env gene of an endogenous murine leukemia virus, elicited a weak Ag-specific T lymphocyte response and resulted in partial protection from challenge with mouse tumors possessing these Ags. Depletion experiments showed that CD8(+), but not CD4(+), T lymphocytes were crucial for the antitumor activity of the vaccines. Systemic administration of agonistic anti-CD40 mAb increased the therapeutic potential of genetic vaccination, but only when given during the tumor rejection phase and not at the time of immunization. This effect correlated with a dramatic increase in the number of ERV-specific CD8(+) T lymphocytes. Adjuvant activity of CD40 agonists thus seems to be relevant to enhance the CD8(+) T cell-dependent response in tumor-bearing hosts, suggesting that sustaining tumor-specific T lymphocyte survival in subjects undergoing vaccination might be a key event in the successful vaccination with weak tumor Ags.

Up-regulation of nuclear protein import by nuclear localization signal sequences in living cells.

Using an in vivo assay system, nuclear import ability in individual cells was determined by examining the nuclear import rate. It was found that when a small (not excess) amount of SV40 T-NLS peptides was co-injected, the nuclear import rate of SV40 T-NLS-containing substrates apparently increased. This up-regulation was reproduced by the co-injection of peptides containing bipartite type NLS of CBP80, but not mutated non-functional NLS peptides, which suggests that these phenomena are specific for functional NLSs. It was further shown that although, in growth-arrested cells, the nuclear import rate was down-regulated compared to growing cells, the elevation of the functional import rate by co-injected NLS peptides reached the same level as in proliferating cells. This up-regulation was abolished by the addition of a protein kinase inhibitor, staurosporine. These results suggest that although potential nuclear import ability does not vary in each cell, the rate of nuclear import may be controlled by the amount of karyophilic proteins, which need to be carried into the nucleus from the cytoplasm, possibly via an NLS-dependent phosphorylation reaction.

Protection of mice against SV40 tumours by Pam3Cys, MTP-PE and Pam3Cys conjugated with the SV40 T antigen-derived peptide, K(698)-T(708).

The intraperitoneal injection of Balb/c mice with synthetic analogues of adjuvants S-[2,3-bis(palmitoyloxy)-(2-RS)-propyl]-N-palmitoyl-R-cysteine (Pam3Cys) or muramyltripeptide phosphatidylethanolamine (MTP-PE) inhibited the tumourigenic growth of subcutaneously injected VLM cells, a syngeneic simian virus 40 (SV40)-transformed cell line. Furthermore, the Pam3Cys conjugate of K698-T708 (KT), which represents the C-terminal undecapeptide of the SV40 large tumour (T) antigen, was tumour-protective. Also syngeneic spleen cells, preincubated in vitro with this Pam3Cys-KT derivative, which anchores spontaneously at the cell membrane, were, through SV40 tumour mimicry, tumour-protective. The protection was impaired by treatment of the mice with either anti-CD4, anti-CD8 IgG, anti asialo GM1 antiserum or dextrane sulfate, which deplete the CD4+, CD8+ and NK cells or the macrophages, respectively. In summary, SV40 tumour transplantation resistance can be experimentally elicited by a tumour-epitope-specific vaccine. In the absence of an immunogenic epitope protection was obtained by administration of biological response modifiers. Protection is effected by SV40-T-antigen-specific cytotoxic lymphocytes in cooperation with NK cells and macrophages.

The nature of antigen in the eye has a profound effect on the cytokine milieu and resultant immune response.

The eye is endowed with a number of mechanisms that protect it from immune-mediated injury. One such mechanism, termed anterior chamber-associated immune deviation (ACAID), evokes the antigen-specific, systemic down-regulation of Th1 responses to antigen inoculated into the anterior chamber of the eye. ACAID has been correlated with the selective production of IL-10 by the antigen-presenting cells (APC) and the development of a cross-regulatory Th2-like response. A small subset of antigens do not induce ACAID, but instead provoke IL-12 and normal Th1 immunity. Remarkably, all soluble antigens tested are capable of inducing ACAID; only cell-associated antigens do not induce ACAID. We hypothesized that the nature of antigen plays a decisive role in the resultant immune response. This hypothesis was tested with two well-characterized antigens, ovalbumin (OVA) and SV40 large T antigen (SV40 Lg T Ag). The soluble forms of OVA and SV40 Lg T Ag induced ACAID in both in vivo and in vitro models of the eye. In contrast, the particulate forms of these antigens, i.e. OVA passively absorbed onto inert latex beads (OVA-latex) and SV40 Lg T Ag expressed in two different cell lines, 99E1 and SV-T2, did not induce ACAID in either in vivo or in vitro models of the eye. In addition, the cytokine profiles of ocular APC pulsed with OVA or OVA-latex showed that soluble OVA induced the production of IL-10, whereas OVA-latex induced the production of IL-12. These data suggest that the nature of the antigen in the eye, whether soluble or particulate, is a crucial determinant in the resultant immune response. Moreover, they suggest a mechanism in which soluble antigens preferentially induce the release of ACAID-inducing IL-10 whereas particulate antigens preferentially induce the release of Th1-inducing IL-12 by responding APC.

T/Tn antigen vaccine is effective and safe in preventing recurrence of advanced breast carcinoma.

Since 1974, and as of March, 1993, we have used T/Tn antigen vaccine in safe, specific, effective, long-term intradermal vaccination against recurrence of advanced breast carcinoma (CA). Staging is by the pathologic TNM system. Treatment is ad infinitum. Of 19 consecutive breast carcinoma patients vaccinated, six Stage IV, six Stage III, and seven Stage II all survived > 5 years postoperatively. Three Stage III, three Stage IV, and five Stage II patients (i.e., 11) survived > 10 to > 18 years. Five others are alive but have not reached 10 years; three of them have no evidence of disease (NED). Three patients died of CA before reaching 10 years. An additional three breast CA patients are being treated for > 2 years, but, < 5 years postoperatively, they are NED. The vaccination are presented as a delayed-type hypersensitivity reaction with significant inflammation with increase of helper T lymphocytes and decrease of T suppressor/cytotoxic cell ratio.

The cell-binding carboxyterminal undecapeptide of SV40 tumour antigen provides protective cell-dependent immunity.

This paper describes the use of the synthetic carboxyterminal undecapeptide of large SV40 tumour antigen, lys698-thr708 (KT) to protect Balb/c mice against growth of subcutaneously transplanted tumorigenic SV40-transformed cells (VLM). The vaccine was prepared by conjugation of KT with 3-(2-pyridyldithio)propionic acid N-hydroxysuccinimide (SPDP). Addition of the SPDP-derivative of KT to syngeneic spleen cells rendered KT covalently linked to free thiol-groups of the cell membranes by the formation of -S-S-CH2-CH2-CO-epsilon-NH-lys698 bonds. Vaccination with KT-conjugated cells was intraperitoneal. Alternatively, KT-conjugated cells were generated in the peritoneum by injection of PDP-KT ((2-pyridyldithio)propionic acid-KT). As a control 60Co-irradiated VLM cells were used. In five experiments all VLM-vaccinated and the majority of the PDP-KT-(or KT-spleen cell)-vaccinated mice were protected against tumour growth. However, mice pretreated with saline, unconjugated spleen cells, free KT, KT conjugated to bovine serum albumin, or KT with incomplete Freund's adjuvant developed tumours. Treatment of PDP-KT-vaccinated mice with anti-CD4 or anti-CD8 immunoglobulin abolished tumour immunity completely. Thus, covalent binding of the carboxyterminal undecapeptide of SV40 tumour antigen to viable, untransformed cells yielded a vaccine which protects Balb/c mice against SV40 tumours.

Oral hairy leukoplakia: a light microscopic and immunohistochemical study.

We compared the light microscopic features and immunoperoxidase staining for human papillomavirus antigen of oral hairy leukoplakia (OHL) and flat condylomatous lesions of the cervix. Our findings support the conclusion that OHL and flat condylomatous lesions of the cervix are not similar morphologically or etiologically. Therefore the term koilocyte seems inappropriate to describe the swollen epithelial cells in OHL. The various diagnostic criteria for a diagnosis of OHL were reviewed. On the basis of the review of our cases of OHL and our review of the literature, we conclude that a definitive diagnosis of OHL requires either the biochemical evidence of Epstein-Barr virus or the ultrastructural demonstration of herpesviruses in suspected lesions.

Recombinant polyoma--vaccinia viruses: T antigen expression vectors and anti-tumor immunization agents.

Live vaccinia virus recombinants expressing viral antigens have recently been developed as effective anti-viral vaccines. We have examined the possibility of extending this approach to specific anti-tumor immunity, using tumors induced by the polyoma virus (PyV) as a model system. Three recombinant vaccinia viruses, separately encoding the three early proteins of the polyoma virus (large, middle and small tumor (T) antigens) were constructed. Each recombinant efficiently expresses the appropriate T antigen, which exhibits biochemical properties and subcellular localization of the authentic PyV protein. The potential of the recombinants to elicit immunity towards PyV-induced tumors was assessed in rats by a challenge injection of syngeneic PyV-transformed cells. After prior immunization with the large-T or the middle-T viruses, small tumors developed, which later regressed and were eliminated in more than 50% of the animals. In contrast, the small-T virus failed to elicit tumor rejection. Established tumors could also be eliminated by curative vaccinations. No circulating antibodies directed against PyV large-T or middle-T antigens were detected in animals vaccinated with the large-T or middle-T viruses, suggesting that rejection may be due to a cell-mediated immune response.

[Induction of chromosome aberrations by the T antigen of the SV40 virus introduced into the cells using liposomes]. / Induktsiia khromosomnykh aberratsii T-antigenom virusa SV40, vvedennogo v kletki pri pomoshchi liposom.

A purified SV40 T antigen introduced into hamster cells by means of liposomes accumulated in the nuclei within 10 h and persisted there further as long as 10 to 12 h. Within the first day after cell treatment with T antigen numerous chromosome aberrations including breaks, translocations and gaps were observed in the cells. The number of aberrations slightly reduced by the 2nd day followed by restoration of the normal cell karyotype by the 5th day. Removal of T antigen incorporated into liposomes by a specific immunosorbent or heat inactivation of T antigen abolished the clastogenic effect. It is suggested that induction of chromosome aberrations might activate sell protooncogenes and thus serve a genetic basis of tumor progression.