B7-1 Gene Research Articles

Effect on biological behavior of chemotherapy-resistant tumor cells by human wild-type p53, GM-CSF and B7-1 genes via recombinant adenovirus

Objective: To explore the effect on biological behavior of chemotherapy-resistant tumor cells by human wild-type p53, GM-CSF and B7-1 genes mediated via recombinant adenovirus. Methods: p53-abnormal KB-v200 (VCR resistant) and KB-s (VCR sensitive) cell lines were used as model tumor cells, which are resistant and sensitive to chemotherapeutic drugs respectively. After infected with recombinant adenovirus carrying human wild-type p53, GM-CSF and B7-1 genes, changes in biological behavior (including drug sensitivity) of these two kinds of gene-transduced cancer cells were observed. Results: Both of the cell lines were susceptible to adenovirus, all of three exogenous genes (p53, GM-CSF and B7-1) could be effectively expressed in these cell lines, their growth was suppressed, and apoptosis was induced. The drug-pumping-out function of Pgp glycoprotein on the cytomembrane of drug-resistant KB-v200 cells was markedly affected 48h after transfection of the recombinant adenovirus, revealed by increase of the amount of rhodamine 123 accumulation in the cells. The MTT assay also indicated the reversal of their sensitivity to VCR drugs.In vivo experiment in nude mice it was demonstrated reduction of tumorigenicity of the KB-v200 cells or KB-s cells infected with the recombinant adenovirus, and increase of their sensitivity to VCR. Conclusion: The clinical application of this recombinant adenovirus carrying agents might be more effective in treatment of tumors with multidrug resistance(MDR).

B7-1 gene-modified autologous tumor-cell vaccines for renal-cell carcinoma.

In recent years, interest in the development of immunologic approaches to malignancies has increased, and there is good evidence that the growth of renal-cell carcinoma (RCC) can be modulated by the host's immune system. Indeed, use of the immunomodulatory cytokine interleukin-2 (IL-2) has been approved for the treatment for this disease. The efficacy of this approach remains low, and there is no other reasonable conventional therapy for patients with metastatic RCC. Therefore, there is a need for the development of novel treatment strategies. The development of autologous tumor-cell vaccines that have been genetically modified to become more immunogenic is an approach that is actively being studied. One of the genetic manipulations that is being employed by several groups is the induction of overexpression of B7-1 to provide costimulation to tumor-reactive T-cells. The rationale for this strategy is that T-cells need two signals before they can mount a cytotoxic response: the binding of the T-cell receptor (TCR) to an antigenic peptide presented on major histocompatibility complex (MHC) molecules and the binding of CD28 to B7-1. Since B7-1 is not normally expressed by RCC cells, the expression forced by transfection of an exogenous B7-1 gene could make the tumor cells more immunogenic. This has been shown to be the case in mice, in which the injection of tumor cells transfected with B7-1 can result in the T-cell-mediated rejection of unmanipulated parental tumor cells. We have applied this approach to the treatment of patients with metastatic RCC. Patients enrolled on our phase I protocol are treated with autologous tumor cells modified to express B7-1, which functions as a tumor vaccine. Primary tumors or metastases are resected from the patients. The tumor cells are adapted to in vitro culture, infected with a recombinant adenoviral vector containing human B7-1 cDNA driven by the cytomegalovirus (CMV) promoter, radiated, and stored in liquid nitrogen. Aliquots of the B7-1 gene-modified tumor cells are given to the patients as a vaccine at varying intervals according to a dose-escalation scheme. The patients also receive systemic IL-2 for the dual purpose of providing accepted therapy for this disease as well as expanding the tumor-reactive T-cells activated by the vaccine. The immunogenicity and toxicity of the vaccine as well as the clinical response are being assessed in three to five patients at each of three dose levels.

Genetic variation in the B7-1 gene in patients with multiple sclerosis

Co-stimulation of T cells by B7-1, a protein that is expressed on antigen presenting cells, favors a T helper type1 (Th1) cellular response. Th1/Th2 bias may influence disease susceptibility or course of MS. We screened the entire coding sequence as well as the 5′- and 3′-untranslated regions of the B7-1 gene using a mutation scanning technique, dideoxyfingerprinting, in DNA from 111 patients with MS from Olmsted County, Minnesota. We identified five genetic variants. None alter protein structure nor have apparent functional significance. Selected variants of sufficient frequency were tested for an association with course and severity of MS and one was tested for an association with susceptibility; none of the association tests were positive.

Molecular characterization of an adenoviral vector resulting from both homologous and nonhomologous recombination.

Recombinant adenoviral vectors are being used extensively for gene transfer. During the construction of an E1-deleted virus expressing the human B7-1 gene, an aberrant recombinant (Ad.ihB7-1) arose with an unusual 5' sequence. Characterization and sequencing of Ad.ihB7-1 showed that its structure was the result of both homologous and nonhomologous events. The most striking features of the construct were the incorporation of bacterial genomic DNA, an additional inverted terminal repeat, and portions of E1a. The appearance of this construct has implications for vector design and indicates the need for careful analysis and characterization of recombinant adenoviral vectors for clinical use.

B7-1 (CD80)-gene transfer combined with interleukin-12 administration elicits protective and therapeutic immunity against mouse hepatocellular carcinoma.

Human hepatocellular carcinoma (HCC) frequently recurs after primary therapy, resulting in poor prognosis. To try to find a way to prevent this, we examined the combined effectiveness of B7-1 (CD80)-gene transfer and interleukin-12 (IL-12) on the induction of protective antitumor immunity against poorly immunogenic BNL1ME A.7R. 1 (BNL) mouse HCC cells. We introduced mouse B7-1 gene into BNL1ME A. 7R.1 cells. Overexpression of B7-1 on BNL1ME A.7R.1 cells resulted in significant inhibititon of subcutaneous tumor development in syngeneic BALB/c mice, but not in complete rejection, suggesting that strong expression of B7-1 molecules may enhance the immunogenicity of BNL1ME A.7R.1 cells in immunocompetent mice. Lymphocyte study revealed that the cytolytic activity generated by immunization with B7-1 transfectants against BNL1ME A.7R.1 cells was mediated mainly by CD8(+) cytotoxic T lymphocytes (CTL). We examined the synergistic effect of IL-12 and immunization with B7-1 transfectants. The combination led to rejection of BNL1ME A.7R.1 cells in 6 of 10 tested mice and delayed tumor development in the remaining mice. Furthermore, the combined treatment against pre-established BNL1ME A.7R.1 tumors resulted in rejection in 3 of 8 tested mice or in significant inhibition of tumor growth in the remaining mice. In vivo lymphocyte subset depletion study indicated that the combined antitumor effect was dependent on the presence of both CD8(+) and CD4(+) T cells. In conclusion, the combination of immunization of B7-1-transfected HCC cells and IL-12 could induce protective and therapeutic immunity against parental HCC cells, and this combination may be therapeutically useful for suppressing recurrence of HCC.

Induced Expression of B7-1 on Myeloma Cells Following Retroviral Gene Transfer Results in Tumor-Specific Recognition by Cytotoxic T Cells

The aim of this study was to evaluate whether tumor cells from patients with multiple myeloma activate allogeneic and autologous T cells. Results showed that myeloma cells expressed few B7-2 and no B7-1 in six cell lines and primary cells from 11 patients. They expressed substantial levels of HLA class I, CD40, and a set of adhesion molecules. In accordance with the low density of B7 molecules on these cells, they were poor allogeneic CD8+ T cell stimulators. Neither IFN-gamma plus TNF-alpha nor CD40 stimulation significantly induced B7-1 or up-regulated B7-2 on human myeloma cell line or primary myeloma cells from six of seven patients. However, such induction was found on autologous bone-marrow nontumoral cells and on autologous dendritic cells following CD40 stimulation. High B7-1 expression was stably obtained on human myeloma cell line using transduction with a B7-1 retrovirus, enabling these cells to stimulate allogeneic CD8+, though not CD4+, T cell proliferation. For one patient with advanced disease, B7-1 gene transfer made it possible to amplify autologous cytotoxic T cells that killed autologous myeloma cells in an HLA class I-restricted manner, but not autologous PHA blasts. These results suggest that B7-1 gene transfer could be a promising immunotherapeutic approach in multiple myeloma.

Experimental study on the antitumor effect of mouse B7-1 gene.

Mouse B7-1 cDNA was cloned by RT-PCR from BALB/C mouse splenic cells and inserted into pcDNA3 to construct an eukaryotic expression vector. This constructor was named pCD-mB7-1, in which the B7-1 cDNA was identified to be consistent with the data from other researchers. pCD-mB7-1 plasmid was transfected into B16(F0) cells, and effective expression of mB7-1 in these tumor cells could be detected till the 6th month by RT-PCR and RNA hybridization. Specific cytotoxity assay of lymphocytes was conducted after culturing with tumor cells and the results demonstrated that B16 cells transfected with B7-1 gene were more effective than B16-wt and B16-neo in inducing specific cytotoxity of lymphocytes against B16-wt cells. It is suggested that expression of B7-1 gene in tumor cells could enhance the immunogenicity and induce the effective antitumor immunity.

Importance of B7-1-Expressing Host Antigen-Presenting Cells for the Eradication of B7-2 Transfected P815 Tumor Cells

We have previously shown that B7-2 (CD86)-transfected P815 tumor cells elicit tumor-eradicating immunity that leads to the regression of the B7-2+ P815 tumor after transient growth in normal DBA/2 mice. Here, we show that both the B7-2 and B7-1 (CD80) molecules contribute to the eradication of B7-2+ P815 tumors as treatment of the mice with both anti-B7-2 and anti-B7-1 mAb was required to prevent B7-2+ P815 tumor regression. The cells that expressed the B7-1 molecule following inoculation of B7-2+ P815 tumor cells into normal mice were not the tumor cells but rather host APCs including MAC-1+ cells present in the draining lymph nodes. Moreover, B7-1-expressing host APCs were found to be important for the rejection of B7-2+ P815 tumors as anti-B7-2 mAb alone, which was ineffective in preventing B7-2+ P815 tumor rejection by normal wild-type mice, was effective in preventing B7-2+ P815 tumor rejection by mice in which the B7-1 gene was disrupted. Finally, consistent with the importance of B7-1-expressing host APCs for the generation of tumor-eradicating immunity against B7-2+ P815 tumor cells, CD4+ T cells (not only CD8+ T cells) were found to participate in tumor-eradicating immunity against B7-2+ P815 tumor cells. Thus, in addition to eliciting tumor-eradicating immunity directly, B7-2+ P815 tumor cells elicit tumor-eradicating immunity indirectly through B7-1-expressing host APCs that present tumor-associated Ags to CD4+ T cells.

Adenoviral transduction of melanoma cells with B7-1: antitumor immunity and immunosuppressive factors.

Previous studies in experimental models have demonstrated that the transduction of human or murine melanoma cells with the co-stimulatory B7-1 molecule induces effective antitumor immune responses. In order to develop B7-1 gene transfer as a therapeutic tool in the clinical management of melanoma, efficient means of in vivo gene transfer must be used. To this end we evaluated in vitro and in vivo immune responses associated with adenoviral transduction of murine and human melanoma cells with B7-1. Adenovirus-mediated transduction of human and murine melanoma cells with B7-1 leads to high-level transgene expression in vitro and in vivo and does not affect MHC class I and II expression. Adenovirus-delivered B7-1 induced antitumor immune responses, on the basis of observations that human melanoma cells transduced to express human B7-1 were able to co-stimulate allogeneic and autologous T cells to proliferate and that murine melanoma K1735 cells transduced to express murine B7-1 were rejected by syngeneic, immunocompetent mice. By contrast, intratumoral injection of an adenovirus encoding murine B7-1 failed to eliminate established murine melanoma (K1735) despite high-level transgene expression in tumor cells. Potent T cell inhibitory factor(s) secreted by both K1735 cells and select human melanoma cells may contribute to the failure to achieve protection in this setting. Thus, immune inhibitory melanoma-derived factors need to be taken into account when considering the clinical use of B7-1 immunotherapy.

A Double Recombinant Adenovirus Expressing the Costimulatory Molecule B7-1 (Murine) and Human IL-2 Induces Complete Tumor Regression in a Murine Breast Adenocarcinoma Model

Tumors that express tumor-specific antigens can maintain growth in an immunocompetent organism. Current hypotheses tend toward T cell anergy as a key component for the inhibition of immunoreactivity against such tumors. Anergy is thought to occur from hyperactive stimulation of the TCR in the absence of costimulation (costimulation leads to proliferation via IL-2 production). Subcutaneous injection of transgenic polyoma middle T transformed breast adenocarcinoma tumor cells (PyMT) in the hind flank of FVB/n mice results in the formation of tumor nodules at this site. We determined the MHC class I and class II, B7-1, and B7-2 expression in the tumor cells by flow cytometry and showed positive staining for only MHC class I. We show that a single E1-deleted adenovirus constructed to express both the costimulatory molecule B7-1 (murine) and human IL-2 genes (Ad5E1 mB7-1/human IL-2) elicits a very potent antitumor response when administered intratumorally. Ad5E1 mB7-1/human IL-2 induced rapid and complete regression (100%) of all tumors compared with Ad5 E1 mB7-1 (38%), Ad CAIL-2 (42%), and Ad5E1 dl70-3 (control vector) (0%). All mice that exhibited complete tumor regression were fully protected in tumor cell challenge experiments. The systemic immunity generated by intratumoral administration of the Ad vectors was associated with a strong anti-PyMT CTL response. These observations indicate that augmenting the immunogenicity of the tumor with coincident expression of B7-1 in combination with IL-2 may prove beneficial in direct tumor immunotherapy.

Study of enhanced immunogenecity of B7-1 gene transfected human hela cell line

Although cervical carcinoma cells may express the human papillomavirus protein E6 and E7, they fail to induce an effective specific cytotoxic T lymphocyte response. Recent studies suggest that expression of CD-80 (B7-1) on tumor cells is effective to induce antitumor immune responses.1,2 In our study, CD-80 gene was transfected into human Hela cell line with a CD-80 expression plasmid (B7-l*pcDNA3) by electroporation, then the immunogenecity of the modified Hela cell was tested in TLMC (tumor lymphocyte mixed culture) system. [3H]thymidine lymphocyte proliferation assays showed that the response of human peripheral blood lymphocytes (PBLS) to CD-80 positive Hela cells demonstrated a substantial increase in cell proliferation compared to the response to control cells. Cocultivation of allogeneic PBLs with CD-80 positive tumor cells for three days can induce an increased secretion of IL-2. Our results demonstrate an immunostimulatory effect of CD-80 expression on cervical cancer cells, which provides a basis for the development of a therapeutic tumor vaccine.

Antitumor effects induced by b7-1 gene modified EL-4 lymphoma cooperated with IL-2in vivo andin vitro

Costimulation plays very important role in T cells activation and tumor immunity. B7-1 is alone of the major costimulatory molecules both in human and rodents. Previous work indicated that B7-1 gene transfected EL-4 lymphoma can induce antitumor immunityin vivo. This paper showed that inoculation of EL-4B7-1+ plus IL-2 could elicit an antitumor effectsin vivo andin vitro. Immunization with EL-4B7-1+ tumor cells or EL-4B7-1+ tumor cells plus IL-2 could significantly prolong the survival time of the EL-4 tumor-bearing mice and also delay the occurrence of the tumor nodein vivo. A strong proliferation response and CTL activity as well as the increased IL-2 and TNF production of the splenocyte from the immunized mice with EL-4B7-1+ or EL-4B7-1+ plus IL-2 were seen in thein vitro TLMC system. These finding indicated that the costimulatory molecule is necessary for inducing an effective antitumor immunity and IL-2 optimal production.

Expression of B7–1 by Pam 212 Squamous Cell Carcinoma Enhances Tumor Cell Interactions with Dendritic Epidermal T Cells but Does Not Affect In Vivo Tumor Growth

Direct antigen presentation of tumor-associated antigens by tumor cells to T lymphocytes may induce clonal anergy as a mechanism of escape from immune surveillance. B7-1 is a costimulatory molecule for the activation of both CD4+ and CD8+ T lymphocytes that prevents the induction of clonal anergy. Thus, the transfer of B7-1 genes into tumor cells can induce protective immunity and lead to tumor rejection of some tumors in model systems of in vivo tumor growth; however, there is no information on whether stable expression of B7-1 can affect the in vivo growth of squamous cell carcinoma, a common skin cancer. Here, we study how the stable cell surface expression of high levels of B7-1 by Pam 212, a murine squamous cell carcinoma, affects tumor cell-lymphocyte interactions (lymphocyte proliferation and cytotoxicity). Consistent with its costimulatory role, we demonstrate that B7-1 can efficiently induce dendritic epidermal T-cell proliferation in three different dendritic epidermal T-cell cell lines. In addition, B7-1 enhances dendritic epidermal T-cell cytolytic activity against Pam 212 cells in an in vitro 51Cr-release assay, which was blocked by CTLA-4/Ig fusion protein. In contrast to dendritic epidermal T cells, the expression of B7-1 does not alter Pam 212 interactions with either cytotoxic T-lymphocytes, natural killer, or lymphokine-activated killer cells. B7-1 expression by Pam 212 cells did not alter its ability to grow tumors in vivo, as their rate of tumor growth was the same as vector-transfected Pam 212 cells, which were B7-1 negative. Our studies indicate that B7-1 gene transfer into Pam 212 does not alter its tumorigenicity, because it does not alter tumor cell-lymphocyte interactions with cytotoxic T lymphocytes, natural killer cells, and lymphokine-activated killer cells. Further studies of B7-1 modified Pam 212 and dendritic epidermal T cells will clarify whether T-cell receptor-gamma/delta-bearing T lymphocytes can play a role in immunotherapy of Pam 212 squamous cell carcinoma.

Interleukin 12 and B7-1 costimulatory molecule expressed by an adenovirus vector act synergistically to facilitate tumor regression.

Stimulation of antitumor immune mechanisms is the primary goal of cancer immunotherapy, and accumulating evidence suggests that effective alteration of the host-tumor relationship involves immunomodulating cytokines and also the presence of costimulatory molecules. To examine the antitumor effect of direct in vivo gene transfer of murine interleukin 12 (IL-12) and B7-1 into tumors, we developed an adenovirus (Ad) vector, AdIL12-B7-1, that encodes the two IL-12 subunits in early region 1 (E1) and the B7-1 gene in E3 under control of the murine cytomegalovirus promoter. This vector expressed high levels of IL-12 and B7-1 in infected murine and human cell lines and in primary murine tumor cells. In mice bearing tumors derived from a transgenic mouse mammary adenocarcinoma, a single intratumoral injection with a low dose (2.5 x 10(7) pfu/mouse) of AdIL12-B7-1 mediated complete regression in 70% of treated animals. By contrast, administration of a similar dose of recombinant virus encoding IL-12 or B7-1 alone resulted in only a delay in tumor growth. Interestingly, coinjection of two different viruses expressing either IL-12 or B7-1 induced complete tumor regression in only 30% of animals treated at this dose. Significantly, cured animals remained tumor free after rechallenge with fresh tumor cells, suggesting that protective immunity had been induced by treatment with AdIL12-B7-1. These results support the use of Ad vectors as a highly efficient delivery system for synergistically acting molecules and show that the combination of IL-12 and B7-1 within a single Ad vector might be a promising approach for in vivo cancer therapy.

Expression of costimulatory molecules B7-1 (CD80) and B7-2 (CD86) on human hepatocellular carcinoma.

Costimulation mediated by costimulatory molecules, such as B7-1 and B7-2, which are ligands for the CD28/cytolytic T lymphocyte associated antigen (CTLA)-4 counter-receptor, plays an important role in the induction of T cell-mediated antitumor immunity. We investigated the expressions of B7-1, B7-2, and human leukocyte antigen (HLA) class I in seven human hepatocellular carcinoma (HCC) cell lines by reverse transcription-polymerase chain reaction (RT-PCR) and flow cytometric analysis. RT-PCR showed that all these human HCC cell lines were positive for B7-1 and B7-2 at the messenger RNA (mRNA) level. Flow cytometric analysis revealed that they all expressed B7-1, B7-2, and HLA class I on the cell surface. However, the expression levels of B7-1 and B7-2 were very low whereas those of HLA class I were high. B7-1 and B7-2 expression could be increased by treatment with interferon alpha and interferon gamma in a dose-dependent manner, although the expression levels of B7-1 and B7-2 after interferon treatment remained low. By transfecting Hep3B cells with a plasmid containing human B7-1 complementary cDNA (cDNA), we were able to establish Hep3B cell lines strongly expressing B7-1. From mixed lymphocytes and tumor cultures analysis, the primary cytolytic activity against parental Hep3B cells could be induced effectively by B71-transfected Hep3B cells. These findings suggested that B7-1 gene transfer is the best way to induce strong expression of this molecule and this might be useful for immuno-gene therapy against human HCC.

Application of interleukin 12 to antitumor cytokine and gene therapy.

In vivo administration of interleukin 12 (IL-12) at 2000 U/mouse induced IL-12-activated killer (IL-12AK) cells in parallel with an elevation in serum interferon-gamma (IFN-gamma) activity. Although NK1.1+CD3- natural killer cells are the major precursor of IL-12AK cells, asialoGM1+CD8+ T-cells were also demonstrated to be novel precursors. Such anomalous killer cells may play an important role in the early stages of the host defense mechanisms against tumors. It was also shown that IL-12 is effective in inducing tumor-specific cytotoxic T-lymphocytes. Consistent with these data, IL-12 had marked activity against various kinds of established tumors when given systemically. Mice cured of tumors by IL-12 treatment acquired tumor-specific T-cell immunity. Moreover, we initially demonstrated that IL-12 was effective in preventing and inhibiting the growth of primary tumors induced by the chemical carcinogen methylnitrosourea using c-Ha-ras transgeneic mice. Finally, we investigated the application of IL-12 to antitumor gene therapy. Transfer of the IL-12 gene into A20 B-lymphoma cells resulted in the continuous production of IL-12 and caused abrogation of in vivo tumorigenicity. Tumor cells transfected with the IL-12 gene are potentially a good tool as a tumor vaccine, as they effectively induced IL-12AK cells, IFN-gamma production, and tumor-specific protective immunity. Although B16-BL-6 melanoma cells, which are a highly metastatic subclone of B16 melanoma cells, showed resistance to IL-12 gene therapy, combination therapy with the B7-1 gene and systemic IL-12 administration almost completely inhibited tumor metastasis. Similar results were obtained using B16-BL-6 melanoma cells transfected with both B7-1 and IL-12 genes. These results suggest that IL-12 is a promising cytokine for antitumor cytokine and gene therapy.

Costimulatory protein B7-1 enhances the cytotoxic T cell response and antibody response to hepatitis B surface antigen.

There is a need for more effective therapy for chronic virus infections. A principle natural mechanism for elimination of virus-infected host cells is activation of viral antigen-specific cytotoxic T lymphocytes (CTL). In an effort to develop methods of inducing virus-specific CTL responses that might be utilized in therapy of virus infections, we have investigated the effect of B7, a costimulatory factor for T-cell activation. In this study we show that delivery of genes encoding human B7-1 and a viral antigen in the same recombinant viral vector to cells of mice induces a greater viral antigen-specific CTL response than does similar delivery of the viral antigen gene alone. Two recombinant adenovirus vectors were constructed with the foreign genes inserted in the early region 3. One of them (Ad1312) directed expression of the surface antigen gene of hepatitis B virus (HBS); the other (Ad1310) directed coexpression of HBS and human B7-1 (CD80) by means of an internal ribosomal entry site placed between the two coding sequences. When inoculated into BALB/c mice, both vectors induced a viral surface antigen-specific CTL response. The response induced by Ad1310 was stronger than that by Adl312 as measured by a chromium release assay for CTL activity and limiting dilution analysis for CTL precursor frequency, indicating that the B7-1 gene co-delivered with the HBS gene had an enhancing effect on the CTL response against surface antigen. Ad1310 also induced a higher titer of antibody against surface antigen than did Ad1312. This result suggests that expression of a costimulatory protein and a viral antigen in the same cells in vivo induces stronger immune responses than expression of the antigen alone. This could be a novel strategy for development of both preventive and therapeutic vaccines against infectious agents.

Expression of MHC Class II and B7-1 and B7-2 costimulatory molecules accompanies tumor rejection and reduces the metastatic potential of tumor cells.

Mouse tumor cells transfected with syngeneic MHC class II genes are highly immunogenic in the autologous host, and induce a potent tumor-specific immunity against wild type tumor. Previous studies with sarcoma tumor cells expressing transfected class II gene products with truncated cytoplasmic domains suggested that during the process of tumor rejection costimulatory molecules are induced on the tumor cells, contributing to the cells' ability to stimulate immunity. In the present study we directly demonstrate that tumor cells containing full-length class II heterodimers are induced to express B7-1 and B7-2 costimulatory molecules during the rejection process. In contrast, tumor cells expressing class II heterodimers truncated for their cytoplasmic tails are not induced to express B7-1 and/or B7-2. Blocking the interaction of the induced costimulatory molecules with their corresponding receptors on T cells prevents tumor rejection. These results support the hypothesis that the cytoplasmic domain of the MHC class II molecule is involved in induction of costimulatory molecule expression, perhaps via intracellular signalling pathways. Because class II, B7 transfected tumor cells are such effective immunogens against ascites and solid tumors, they have also been tested in metastatic disease. K1735 and B16BL6 mouse melanomas, when transfected with syngeneic MHC class II and B7-1 genes, are significantly less metastatic than parental cells, and immunization with the transfectants protects against subsequent challenge with wild type tumor.

Constitutive expression of B7-1 (CD80) on mouse keratinocytes does not prevent development of chemically induced skin papillomas and carcinomas.

Expression of the B7-1 (CD80) costimulatory molecule in a variety of tumor cell lines leads to an enhanced CD8+ T cell response to tumor Ags. We used transgenic mice constitutively expressing B7-1 on keratinocytes (K14/B7-1 line) to determine whether keratinocyte B7-1 expression would inhibit the development of papillomas and carcinomas following two-stage chemical carcinogenesis in skin. FVB inbred mice carrying the K14/B7-1 transgene and controls were initiated with 25 micrograms of 7,12-dimethylbenz[a]anthracene and promoted weekly with 5 micrograms of 12-O-tetradecanoylphorbol-13-acetate for 20 wk. Expression of the B7-1 transgene did not result in statistically significant decreases in the mean number of papillomas or carcinomas compared with controls. The incidence of carcinomas in both transgenic and control mice reached 90% or greater by 60 wk after initiation. Carcinoma cell lines established from the K14/B7-1 mice maintained expression of B7-1 and Kq. These B7-1 expressing carcinomas grew progressively following intradermal injection into syngeneic FVB mice, further demonstrating their inability to evoke protective tumor immunity. These same carcinoma cell lines were rapidly rejected by minor alloantigen-mismatched SWR mice, confirming their susceptibility to immune effector mechanisms. The failure of constitutive B7-1 expression on keratinocytes to prevent the growth of squamous cell papillomas and carcinomas may reflect the limited immunogenicity of tumors arising after initiation-promotion carcinogenesis. Our results in this transgenic model system are further evidence that B7-1 gene therapy alone may not be sufficient to induce protective immunity to some types of tumors.

B7-1 gene transfer into human cancer cells by infection with an adenovirus-B7 (Ad-B7) expression vector

Transfection of the costimulatory molecule B7-1 into some murine tumors can increase antitumor immunity and eradicate tumor growth. The purpose of this work was to construct an adenovirus-B7 (Ad-B7) expression vector and study B7-1 gene transfer into human cancer cells. The human B7-1 cDNA was ligated into an expression cassette containing the human cytomegalovirus immediate early gene promoter and then inserted into the E1 region of the Ad5 genome by homologous recombination. The resulting Ad-B7 vector was used to infect established cancer cell lines and freshly resected cancers. Resected tumors were disaggregated into single cell suspensions by mechanical mincing and enzymatic digestion. Surface expression of B7-1 after infection was verified by flow cytometry. Expression kinetics in three cell lines showed that infected cells began to express B7-1 within 24 h. The proportion of B7-1+ cells continued to increase during the next 48 h, after which expression remained relatively constant during the next 5 days (up to 98% B7-1+ cells). Fresh tumor cells from various cancers displayed similar kinetics, but with greater variability in the proportion of cells expressing B7-1 (13% to 95% B7-1+ cells). Cancers which were successfully infected included 3 colorectal adenocarcinomas, 2 leiomyosarcomas, 2 lung squamous cell carcinomas, and 1 renal cell carcinoma. The Ad-B7 vector is a rapid and efficient means of gene transfer which does not require host cell proliferation. The ultimate objective is to engineer autologous tumors to express B7-1 and vaccinate cancer patients in an adjuvant or palliative setting.