Enhanced immunogenicity has been reported following transfection of a variety of immunogenic tumors with the B7.1 co-stimulatory molecule. The purpose of the present study was to determine if transfection of a weakly immunogenic rat brain tumor, the F98 glioma, with the gene encoding B7.1 could enhance its immunogenicity. F98 cells were transfected with a plasmid containing the B7.1 gene, and stable transfectants (F98/B7.1) were obtained. Flow cytometric analysis confirmed the expression of B7.1 and MHC class I antigens on the cell surface. To investigate the effects of B7.1 expression on the tumorigenicity of the F98 glioma, Fischer rats were implanted intracerebrally with either F98 (wild-type) or F98/B7.1 transfected cells. No significant differences in survival times were noted. Mean survival times of 21.8 and 24.0 days were observed for the respective groups at a challenge dose of 103 cells. These differences in survival time were not significant. To determine if expression of B7.1 enhanced the immunogenicity of the F98 glioma, rats were vaccinated weekly for 3 weeks with 107 mitomycin C-treated F98 or F98/B7.1 cells injected subcutaneously and then challenged intracerebrally with F98 cells 1 week later. Unvaccinated animals or those that received wild-type F98 cells as a vaccine had a survival time (mean ± s.d.) of 22.3 ± 1.5 days following tumor challenge versus 20.0 ± 1.7 days for rats that had been vaccinated with F98/B7.1. Although we recognize that it might be possible to design more effective vaccination regimes, nevertheless, our data indicate that transfection of the B7.1 gene into the F98 rat glioma did not enhance its immunogenicity, and that other approaches will be required.
Antigen specific T cell responses occur following T cell receptor (TCR) interaction with a peptide antigen in association with the MHC on the surface of an antigen presenting cell (APC). However, the TCR/MHC-Ag interaction by itself is insufficient to activate naive T cells,1 and a second costimulatory signal is required.2 In the absence of this, not only will naive T cells not be activated, but a state of T cell unresponsiveness will be induced, rendering T cells incapable of future activation against the presented antigen. It is now well recognized that one source for this second signal is the interaction of the CD28 and/or CTLA4 molecules on the surface of responding T cells with the B7.1(CD80) and B7.2(CD86) molecules on the surface of APCs.123
Most tumors do not express these costimulatory molecules, which may account for their weak immunogenicity. Despite the presentation of potentially immunogenic tumor antigens in the context of the MHC, the lack of costimulatory molecules may prevent the activation of the T cells necessary for an effective anti-tumor response. It has been reported that transfection of genes encoding costimulatory molecules into tumor cells, can enhance their immunogenicity,4567 and data derived from several animal tumor models support this.456891011 Using murine melanoma cell lines expressing the E7 gene product of human papillomavirus 16 as a model tumor antigen, Chen et al4 have observed immune rejection of E7+ tumors when transfected with B7.1, but not of either E7− or B7.1− tumors. Furthermore, following immunization with the transfected tumor cells, mice rejected E7+B7.1− tumors at a site distant from the primary tumor. Townsend and Allison6 have reported that the immune rejection of B7.1 expressing murine melanomas can occur in vivo against endogenously encoded tumor antigens presented on the MHC class I molecules without the expression of a xenoantigen. Similar results have been reported using a variety of murine tumors including lymphoma and mastocytoma cell lines,512 a murine sarcoma in which tumor rejection was found to be mediated by CD4+ T cells,8 and plasmacytoma and thymoma cell lines.910
High grade gliomas, specifically glioblastoma multiforme (GBM) and anaplastic astrocytomas (AA), are extremely resistant to all current forms of therapy, including surgery, chemo-, radioimmuno- and gene therapy. Despite aggressive therapy, overall survival of individuals diagnosed with GBM is less than 2% and the mean survival time following diagnosis is approximately 18 months.12 These dismal survival statistics have provided the impetus to develop innovative therapeutic strategies, including gene therapy. GBM and AA are both locally invasive and diffusely infiltrative, initially in the cerebral hemisphere of their origin, but with recurrence and progression the contralateral hemisphere may be involved. The challenge for the future is to devise strategies that will be effective for treating both primary and recurrent tumors. Immunotherapy and gene therapy potentially may be useful adjunctive treatments but, as outlined in a recent review by Shapiro,13 significant problems must be overcome. Although there are few reports of direct attempts to increase the immunogenicity of brain tumors, a number of studies on gene therapy of brain tumors have reported enhanced tumor immunogenicity following transfection with the herpes simplex thymidine kinase (HStk) gene followed by treatment with ganciclovir.1415 Previous studies with the F98 rat glioma have demonstrated that this tumor line resembles human glioma in its biologic behavior and lack of response to chemo-, radio-, and immunotherapy.1617181920 Using the weakly immunogenic F98 glioma as a model for human GBM, the purpose of the present study was to determine if expression of B7.1 by the F98 glioma could provide the necessary costimulatory signals for T cell activation and thereby lead to a protective immune response against this tumor, which until recently,19 has been incurable.
Transfected cells express B7.1 and MHC class I on their surface
The cell surface expression of B7.1 and MHC class I and class II molecules on F98 wild-type and transfected cells was determined by flow cytometry. B7.1 was expressed on F98 cells transfected with the plasmid encoding B7.1, but not wild-type or vector control cells (Figure 1). The relative fluorescence intensities of isotype-stained controls were equivalent to one another and to B7.1-stained, unmodified F98 cells. MHC class I, but not class II molecules were expressed on F98 wild-type cells, and the level of expression, as measured by the mean channel of fluorescence (MCF), was minimally affected by transfection with the gene encoding B7.1 (F98 wt: MHC I MCF = 9.9, MHC II MCF = 1.0; F98/B7.1: MHC I MCF = 12.7, MHC II MCF = 0.8; Figure 2). Wild-type or transfected F98 cells were seeded into six-well tissue culture plates at a density of 105 cells per well. At approximately 24-h intervals, cells were harvested and counted in triplicate and the cell counts averaged to determine the in vitro growth rate. Although there were minor differences in doubling time between the wild-type (16 h) and transfected cells (16–18 h), these differences were not statistically significant.
Tumorigenicity of transfected F98 cells
Once it had been determined that the transfected F98 cells expressed B7.1 and that their growth was not affected by transfection, experiments were designed to determine what, if any, effect the expression of B7.1 had on the in vivo growth of the F98 cell line. Fischer rats were implanted intracerebrally (i.c.) with 103, 104, or 105 wild-type or transfected F98 cells. The survival data are shown in a series of Kaplan–Meier plots (Figure 3), and mean survival times (MST) and median survival times (MeST) are summarized in Table 1. Log-rank analysis of the survival data, stratified according to tumor challenge dose, failed to reveal any significant differences (F98 wt versus F98/B7.1, P = 0.32; F98 wt versus F98/pRC-CMV, P = 0.23) in survival times for rats implanted with F98 wild-type cells and the various transfectants.
Response of vaccinated rats to tumor challenge
Once it had been determined that B7.1 expression by the transfected F98 cells did not alter their tumorigenicity, experiments were initiated to determine if they had enhanced immunogenicity. To test this hypothesis, rats were injected weekly for 3 weeks with either 107 wild-type or transfected F98 cells. On the fourth week, they were challenged with either an i.c. implant of 104 F98 cells or a s.c. injection of 107 F98 cells to determine if vaccination had evoked an immune response against the glioma. To provide a positive control for our vaccination protocol, additional Fischer rats were vaccinated and challenged with the strongly immunogeneic 9L rat gliosarcoma using the same procedure, since it previously has been demonstrated that this regimen was effective in conferring immunity.2122
The growth rates of subcutaneous (s.c.) implants of F98 tumors were similar in rats that had been subjected to an immunizing regimen with F98/B7.1 cells versus wild-type cells (Figure 4a). As the s.c. tumors grew, several of them developed necrotic areas, leading to greater variability in tumor volumes within each group, although the mean tumor volumes for each group were similar throughout the study. On the other hand, tumor growth in rats immunized and challenged with the 9L gliosarcoma (Figure 4b) was much slower in immunized versus unimmunized rats and the tumor volume was smaller (2.2 ± 1.5 cm3 versus 0.8 ± 0.7 cm3, respectively) at day 10. Furthermore, the 9L tumors regressed rapidly until they became undetectable in 9L immunized rats, while 9L tumors in unimmunized rats grew faster and persisted for a longer time period. Ultimately, however, these tumors also regressed and eventually became undetectable by day 45.
Survival data for rats receiving i.c. challenges with F98 or 9L gliomas are shown in Figure 5 and are summarized in Table 2. MeST for the rats following i.c. implantation of the F98 tumor ranged from 20 to 22 days. Although there was a statistically significant difference (F98 wt-vaccinated versus F98/B7.1-vaccinated, P = 0.02) in survival times between the transfectant-vaccinated groups and the controls (Table 2), the actual differences in MST and MeST are only 1 to 2 days, and therefore were not biologically significant. This point becomes more evident when these results are contrasted with the results of 9L vaccinated and challenged animals, where all five unimmunized animals challenged with the 9L glioma died between 28 and 37 days after implantation (MST = 33, MeST = 34) compared with 60% long-term survival of immunized animals (Figure 5b, P < 0.002, log-rank statistic).
In the present study, we have demonstrated that expression of the B7.1 co-stimulatory molecule by the F98 glioma did not enhance its immunogenicity. Although B7.1 was expressed by F98 glioma cells, the transfected cells grew at the same rate, and resulted in the death of tumor-bearing rats at the same time following implantation as wild-type cells, indicating that the tumorigenicity was unaffected by gene transfection. Furthermore, when transfected cells were used as a vaccine before either s.c. or i.c. challenge with the wild-type tumor, there was no effect on tumor growth (s.c.) or survival time (i.c.) of the animals.
Previous studies on the immunotherapy of brain tumors have shown that vaccination with an immunogenic tumor is an effective method of preventing experimental tumor engraftment and of treating established experimental tumors.2122232425 One of the earliest examples was the report by Blume et al21 in which rats were immunized with irradiated 9L cells before either a s.c. or i.c. challenge of 9L cells. In both the i.c. and s.c. challenge groups, tumors failed to grow in the immunized animals, while unimmunized animals developed progressively growing tumors. Denlinger et al22 reported similar results using the T9 tumor, which in reality was the same tumor as the 9L gliosarcoma.20 We observed similar results with the 9L tumor using a less intensive vaccination regimen.
We chose to use the F98 glioma for this study, since its in vivo biologic behavior closely resembles human GBM in that it diffusely infiltrates surrounding brain and is weakly immunogenic.1718 This is especially important, since the majority of experimental studies on the immunotherapy of brain tumors have employed tumors that are highly immunogenic.2122232425 This is in contrast to human brain tumors, which appear incapable of evoking an effective immune response.26 It has been convincingly demonstrated that introduction of the genes that encode B7.1 and/or B7.2 into tumor cells can improve antitumor immunity.456789101112 Although these studies have demonstrated enhancement of tumor immunogenicity following B7.1 transfection, we were unable to show a similar effect with the F98 glioma. There are several possible explanations for this. First, the level of expression of B7.1 expression by the tumor cells may have been insufficient to provide the necessary costimulatory signal.27 It has been hypothesized that low levels of B7.1 expression by resting APC results in the preferential engagement of the CTLA-4 receptor on T cells thereby inhibiting activation.3 It is only when B7.1 is expressed at higher levels following the activation of APC that CD28 molecules are engaged and the T cells activated. Second, it is possible that the human B7.1 molecule was not sufficiently cross-reactive with its rat homologue. Although there is no direct evidence of cross-reactivity, we consider this possibility to be unlikely. The coding sequences of rat, mouse and human B7.1 genes have a high degree of homology (80% and 67%, respectively)28 and of the 20 amino acids identified as most important for B7.1/CD28 interaction,2930 18 are conserved between rat and human sequences.28 Furthermore, human and murine B7.1 have been proven to be functionally interchangeable,711 despite less conservation of the amino acids involved in CD28/B7.1 binding. Finally, the rat B7.1 molecule has been demonstrated to interact functionally with the human CD28 and CTLA-4 molecules.2831 All of these observations strongly support the contention that human B7.1 will functionally interact with rat CD28 and/or rat CTLA-4. Third, it is possible that F98 cells may have been incapable of presenting antigen to the naive T cells due to the lack of MHC II on the tumor cells surface. However, when B7.1 expressing tumor targets only express MHC I, they can be demonstrated to directly activate the CD8+ T cells.6 Furthermore, previous studies have shown that an immune response to the F98 can be evoked following vaccination,1718 despite a lack of detectable MHC II on the cell surface.
Our data support those recently reported by Visse et al32 who were unable to induce immunity using a B7-expressing tumor cell vaccine to cure male rats bearing i.c. implants of the N32 rat glioma. They were, however, able to cure a majority of immunized female rats. They postulated that the female rats may have mounted a stronger tumor-associated immune response than male rats, or alternatively that the immune response may have been directed against weak, H-Y antigens expressed on the N32 tumor, which originated in a male rat.33 Our data support the second hypothesis, since repeated immunizations of female rats failed to evoke an immune response against the F98 glioma. Karyotypic analysis (Theil K and Barth R, unpublished data) indicate that the F98 glioma arose in a female rat since it lacks a Y chromosome. This hypothesis is further supported by the data of Tzeng et al1718 showing that a small, but significant increase in both survival and cytolytic cell precursor frequency was observed when male rats were vaccinated with the wild-type F98 tumor. Our results also extend those of Visse et al by defining the effects of B7.1 expression on the tumorigenicity of an intracerebral glioma. We demonstrated that not only does transfection of B7.1 not improve the immunogenicity of this tumor, but it also has no effect on its tumorigenicity. It is interesting to note that in previous studies, even when it was not possible to generate protective immunity to weakly immunogenic tumors, tumorigenicity was reduced when B7.1 was expressed.2734
Whether or not H-Y antigens are involved, the difference is most likely in the degree of immune response generated to the wild-type tumors. The ability of the wild-type tumor to evoke an immune response appears to be crucial in order for B7.1 expression to enhance an antitumor immune response.27343536 Although other investigators have reported effective augmentation of antitumor immunity following transfection of tumor cells with B7.1,456891011 many of these studies employed immunogenic tumors. When the wild-type N32 glioma was used as a vaccine, an immune response was raised, demonstrating the existence of tumor rejection antigens. The response to these antigens, whether tumor-specific or gender-related, was then improved in the female rats following the expression of B7. In our study we were unable to induce any immunity with wild-type F98 glioma cells suggesting that no tumor antigens were present, and thus there was no immune response to improve. Furthermore, since there was not even a modest survival increase in survival time following immunization with the wild-type tumor, it is not surprising that B7.1 should have no effect. Since most human brain tumors are weakly or non-immunogenic, our studies suggest that B7.1 transfection alone may not be a successful therapeutic strategy, and that other approaches will be required.
Materials and methods
Construction of B7.1 vector and transfection of F98 glioma
A cDNA of the entire coding region for human B7.1 was constructed from mRNA of a K562 erythroblastoid human leukemia cell line by reverse transcription and PCR amplification using specific primers. The oligomer primers contained sequences for directional insertion into the pRC-CMV plasmid vector (Invitrogen, San Diego, CA, USA) at the 5′ and 3′ ends, which could be cleaved by the restriction endonucleases HindIII and XbaI (Boehringer Mannheim, Indianapolis, IN, USA), respectively. Gel electrophoresis of the PCR product confirmed a cDNA with the expected size of 954 bp (data not shown). This cDNA was isolated, incubated with HindIII and XbaI and ligated into the pBluescript plasmid, which was then used to transform E. coli. Transformed bacteria were lysed and the plasmid DNA was isolated using a previously described method.37 The B7.1 cDNA was sequenced from the isolated plasmid using the dideoxy method38 and the pBluscript-specific primers T7 and KS and found to be free of point mutations. The insert was then cut out of pBluescript and sub-cloned into the pRC-CMV plasmid, which was grown in large scale to obtain purified plasmid DNA for transfection into the F98 rat glioma cell line (American Type Culture Collection, Manassas, VA, USA; No. CRL-2397).
Transfection of the pRC-B7.1 into F98 glioma cells394041 was carried out in semi-confluent T-25 flasks in serum-free Optimem medium (Gibco BRL, Gaithersburg, MD, USA) during exponential growth phase. The Lipofectin (Gibco BRL) reagent was mixed with 1 μg of plasmid DNA and kept at room temperature for 15 min following which it was overlaid on the cells and incubated at 37°C and 5% CO2 for 24 h. The cell monolayer was then rinsed twice with cold Hanks’ solution and re-incubated with DMEM media with 10% fetal bovine serum. After 48 h, the DMEM was supplemented with neomycin 200 μg/ml for selection and the cultures monitored daily using an inverted microscope, subcultured and media changed as necessary.
Subconfluent monolayer cultures of both wild-type and transfected F98 cells were disaggregated with cold 1 mM EDTA and then the cell suspension was vigorously pipetted several times to further dissociate any remaining cell clumps. The cells were washed once in phosphate buffered saline (PBS), pH 7.4 and aliquots containing 5 × 105 cells were dispensed into 12 × 75 mm glass tubes for staining. To determine if B7.1 (CD80) was expressed on the transfectants, cell pellets were resuspended in 20 μl of a FITC-conjugated mouse anti-human CD80, or a FITC-conjugated mouse IgG1 isotype control antibody (Becton Dickinson, San Jose, CA, USA). Cells were placed on ice for 20 min and then washed once with PBS. To determine MHC expression, cells were resuspended in 20 μl of a 1:500 dilution of ascites containing either monoclonal mouse anti-rat-MHC class I, monoclonal mouse anti-rat-MHC class II, or mouse IgG1 isotype control antibody (Harlan/Serotec, Indianapolis, IN, USA). Cells were held on ice for 20 min, and then washed once with PBS. Cell pellets were then resuspended in 20 μl of a 1:50 dilution of FITC-conjugated goat-anti-mouse antibody (Sigma, St Louis, MO, USA) and again held on ice for 20 min after which they were again washed once with PBS. Following the last washing, the cells were resuspended in 200 μl of 1% paraformaldehyde in PBS, and held at 4°C until flow cytometric analysis was performed. Cells were analyzed for forward light scatter versus log 90° light scatter using a Coulter Elite flow cytometer and electronic gating was used to exclude cellular debris. Green fluorescence was then measured and plotted on a logarithmic scale.
Vaccination and tumor challenge
Wild-type and pRC-B7.1 transfected F98 tumor cell lines, which had been propagated in vitro, were disaggregated with cold (4°C) 1 mM EDTA in PBS. Cells were resuspended in DMEM (1–3 × 107 cells/ml) containing 0.1 mg/ml of mitomycin C (Sigma) and incubated at 37°C for 1 h, following which they were washed four times with PBS. The cells were then resuspended in DMEM at a concentration of 1 × 108 cells/ml. Female Fischer 344 rats (Harlan, Indianapolis, IN, USA) weighing 170–190 g were injected s.c. with 100 μl of the cell suspension s.c., in the nuchal region, directly behind the head. Excipient control rats received s.c. injections of sterile DMEM only.
For s.c. tumor challenges, F98 wild-type and transfected F98 cells, or 9L rat gliosarcoma cells, which had been propagated in vitro, were disaggregated with cold (4°C) 1 mM EDTA in PBS, washed and resuspended in unsupplemented DMEM at a concentration of 108 cells/ml. Tumor cell suspensions (107 cells/100 μl) were injected s.c. into the shaved right flanks of Fischer rats. Tumor growth was assessed by measuring their dimensions with calipers in two orthogonal coordinates (c and d), and the average diameter was then determined and used to calculate tumor volume using the formula V = 0.52 [(c + d)/2]3.
Intracerebral tumor challenge
F98 wild-type, transfected F98, or 9L gliosarcoma cells were stereotactically implanted i.c. into the right caudate nucleus of Fischer rats using a procedure described in detail elsewhere.1619 Rats were weighed at least three times per week to monitor their clinical status. As determined from previous studies with the F98 glioma,151619 the combination of sustained weight loss, ataxia and periorbital discharge indicated that death was imminent. Therefore, to minimize discomfort, animals displaying these signs were killed. Survival times were determined from the day of implantation to the day of death plus 1 day. Kaplan–Meier survival plots were prepared and log-rank analysis was performed on all survival data to determine statistical significance.
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David B Paul was supported by the National Cancer Institute, National Research Service Award CA-09338, Division of Cancer Prevention and Control and the research was supported by a Board of Regents Targeted Interdisciplinary Seed Grant, Office of Research, The Ohio State University and The Ohio State University Comprehensive Cancer Center.
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