Efficient generation of cytotoxic T lymphocytes against cervical cancer cells by adeno-associated virus/human papillomavirus type 16 E7 antigen gene transduction into dendritic cells

2.1 AAV/E7/Neo-transduced DC express E7

The goal of this study was to observe whether AAV-based antigen gene delivery into DC was able to elicit a significant CTL response. Fig. 1A shows a structural map of the AAV/E7/Neo vector used in this study. In this vector, the E7 gene was expressed from the AAV p5 promoter, which is known to be active in DC 15. A two-step process was used to generate an AAV/E7/Neo virus stock, as previously described 15. However, we found that generating producer cell lines for AAV/E7/Neo was more difficult than for other AAV vectors. Sometimes the clones would spontaneously die (possibly apoptosis). Fig. 1B shows a comparison of various G418 resistant producer cell lines for their ability to produce AAV/E7/Neo virus. Fig. 1C shows that the titer of the virus stock, in encapsidated genomes per milliliter (eg/ml) was about 10⁹ eg/ml.

Protocols for the generation of DC by differentiation of peripheral blood Mo usually involve treating adherent Mo with GM-CSF and interleukin-4 (IL-4). We have modified these protocols in order to promote AAV vector transduction in DC 15 precursor Mo by treating adherent Mo with GM-CSF alone for several days before the addition of IL-4 on day 3. This will allow a brief period of Mo proliferation, which will promote a higher level of AAV transduction. A schematic diagram of the experimental protocol is shown in Fig. 1D. On day 3, after AAV infection and GM-CSF treatment, the cells were finally treated with IL-4 to induce differentiation into DC. The transduction of the Mo/DC population was first observed by measuring poly-adenylated RNA expression of the E7 transgene. At day 5, polyadenylated RNA was isolated from AAV/E7/Neo-infected and mock-infected DC cultures (after IL-4 introduction and differentiation into DC). This mRNA was then analyzed for E7 expression by reverse transcriptase-polymerase chain reaction (RT-PCR). A cellular gene, TF_IIB, was also undertaken as a control. As shown in Fig. 2, E7 expression was only taking place in the virally infected DC.

2.2 AAV delivery results in high levels of antigen-positive DC

The efficiency of virus/gene and lipofectin/protein-pulsing of Mo/DC was analyzed and compared by intracellular staining. Our initial attempts at analyzing E7 levels were unsuccessful. We therefore utilized an equivalent AAV/E6-E7/Neo vector under the same conditions and analyzed for E6 protein levels. The cells were analyzed at day 7 of the protocol. The results, shown in Fig. 3, demonstrate that AAV/E6-E7/Neo infection of Mo resulted in a higher percentage of cells containing intracellular E6 protein.

2.3 AAV/E7/Neo infection results in chromosomal integration

We next observed chromosomal integration of the AAV/E7/Neo vector in DC. Chromosomal integration, while not essential for gene expression from AAV vectors, does signify a permanent genetic alteration of the DC, and is a desirable "gold standard" for viral transduction. The analysis was carried out by PCR amplification of vector-chromosome junctions using primers complementary to the simian virus 40 (SV40) promoter within the vector and Alu I repetitive chromosomal elements 15. AAV-cell junction products were analyzed by PCR amplification and Southern blot analysis, and probing for the Neo gene sequences. Multiple vector-chromosomal junction products were observed in the AAV/E7/Neo-infected DC, but not in mock-infected DC (Fig. 4). These data indicate that at least a subset of the viral genomes are able to chromosomally integrate in the DC population.

2.4 AAV/E7/Neo-transduced DC primed and propagated E7-specific CTL

The ability of the AAV/E7/Neo vector to generate CTL was next analyzed. DC were pulsed by three different techniques as shown in Fig. 1D. One interesting nuance observed during the priming process was the formation of cell rosettes or clusters in the AAV/E7/Neo-pulsed cultures 2 days after addition of the T cells (Fig. 5). By studying the morphology of individual cells within these rosettes, we believe they may represent DC-T cell rosettes and may be an evidence of enhanced priming. Similar DC-T cell rosettes or clusters have been reported by others 27–29. To determine the ability of the AAV/E7/Neo-transduced DC to prime and propagate E7-specific CTL, we carried out a standard 6-h ⁵¹Cr-release assay on day 7, using the T cell populations in the co-cultures. Fig. 6A and B show that the T cell population derived from co-culture with AAV/E7/Neo-transduced DC was able to lyse HLA class I-matched E7-positive primary cervical tumor cell lines, indicating the ability of AAV/E7/Neo-transduced DC to prime and propagate E7-specific CTL. When we analyzed CTL generated from co-culture with E7 protein or lysate-pulsed DC, CTL primed and generated with the use of AAV/E7/Neo-transduced DC were able to mediate target lysis in a much more efficient manner. HLA class I-restricted target lysis was further confirmed by the ability of a monoclonal antibody directed at the monomorphic HLA class I molecules to block killing. Tumor cell lysis was also not mediated through NK cells because no cytolysis was observed when K562 cells were used as targets. To further address the ability of this technique to specifically recognize the E7 target antigen we performed an addition cytotoxicity assay. In this experiment the target cells were autologous lymphoblastoid cell lines (LCL) either unmodified, modified by the lipofection of GST-E7 protein, or infected with AAV/E7/Neo virus. The primed T cells were generated as shown in Fig. 6A and B. As shown in Fig. 6C, with the use of this fully autologous system, the primed T cells had little activity (10%) against unaltered self-LCL, moderate activity in killing GST-E7-lipofected LCL (25%), and strong activity against AAV/E7/Neo-infected LCL (86%) (average of two experiments). These results confirm the antigen specificity of this technique.

2.5 Immunophenotypes of T cells

Immunophenotyping of the AAV/E7/Neo-pulsed DC-primed T cell population showed that this T cell population expressed predominantly CD8 (Fig. 8) and that the CD8:CD4 ratio was slightly higher than in the protein-pulsed and primed situation. In addition, AAV/E7/Neo-pulsing resulted in only low levels of CD56 marker expression. In sharp contrast, pulsing by the lipofection of GST-E7 resulted in much higher levels of CD56. Although CD56 is generally believed to be a natural killer T cell-specific marker, HLA class I-restricted CD8⁺ T cells can also express CD56 30.

2.6 Cytokine profile of T cells

To determine the cytokine profile of the T cells generated from co-culture with AAV/E7/Neo-transduced DC, we carried out intracellular staining of these T cells for IFN-γ and IL-4. Fig. 8 demonstrates that most of the T cells expressed IFN-γ and very little IL-4, suggesting that these cells are of the Th1 phenotype. A smaller proportion of IFN-γ-producing T cells were observed in the T cell populations co-cultured with either E7 protein or tumor lysate-pulsed DC.

2.7 Analysis of DC by various manipulations

Finally, we characterized the DC resulting from the various pulsing techniques to observe whether significant differences were discernible. We used flow cytometric analysis to determine the phenotype of untreated, lysate-pulsed, GST-E7-pulsed, and AAV/E7/Neo-pulsed DC populations. The results, shown in Figs. 9 and 10, demonstrate that the DC generated from all four techniques share all of the common DC markers. However, AAV/E7/Neo-pulsed DC did express significantly higher levels of CD80 and CD83, and slightly lower levels of CD86 than those produced by GST-E7-pulsed DC.

4.1 Materials

The primary cervical cancer cell lines, CA1 (patient 1) and CA2 (patient 2), have previously been described 12. Both contain HPV-16 DNA and were approximately ten passages from initial isolation when used in this study. The cells were grown in Keratinocyte-SFM supplemented with epidermal growth factor and bovine pituitary extract (Gibco BRL/Life Technologies). Both cell lines contain the HLA-A1 haplotype. Mo and DC were derived from peripheral blood mononuclear cells (PBMC). PBMC were obtained from a normal donor with the HLA-A1 haplotype. The lymphoblastoid cellline was generated by transforming donor B cells with Epstein-Barr virus (EBV) as previously described 12.

4.2 Construction of the rAAV/Ag genomes

The AAV/E7/Neo and AAV/E6-E7/Neo genomes were constructed in a similar strategy to that previously described AAV/GM-CSF/Neo viral genome 15. However, instead of the GM-CSF gene, the HPV-16 E7 and E6-E7 open reading frames were cloned by PCR amplification using Pfu polymerase and ligated into the vector, respectively. High-titer rAAV stocks were generated in a two-step process using the complementor plasmid ins96–0.8 and titered as described previously 15, 33.

4.3 Generation and infection of Mo/DC

The PBMC were inoculated into six-well culture plates for 2 h at 37 °C and 5% CO₂, and the adherent cells were harvested following three gentle washes. Immediately after the removal of the nonadherent cells, the adherent Mo were infected with 0.5 ml of virus stock (10⁹ eg/ml). After 2 h of incubation the medium/virus solution was removed, and the cells were washed and replaced with fresh AIM-V medium. Altogether, the Mo/DC precursors were infected with the virus stock three times: on days 0, 3, and 5. GM-CSF (LEUKINE^®, Immunex Corporation) at a final concentration of 800 IU/ml was included in the medium throughout the culture. To induce the maturation of Mo into DC, human IL-4, (R&D SYSTEMS Co.) at 1,000 IU/ml was added at day 3.

4.4 Generation of autologous LCL targets

Donor LCL (2×10⁶) were lipofected with 50 μg of GST-E7 protein 1 day before the ⁵¹Cr release assay to generate one type of autologous target. Donor LCL (2×10⁶) were infected with 0.5 ml of the virus stock 3 days before the ⁵¹Cr-release assay to generate a second type of autologous target.

4.5 Generation of bacterial E7 and E6 protein-pulsed Mo/DC

Mo/DC were generated as above and pulsed with 50 to 100 μg of GST-E7 protein on day 5 as previously described 12.

4.6 RT-PCR analysis

E7 mRNA expression was detected by RT-PCR amplification along with a cellular mRNA control. Total RNA was isolated Trizol reagent (Gibco BRL Life Technologies Inc.) and treated with 5 U/μgof RNase-free DNase I (Promega Co.) at 37°C for 2 h. Messenger RNA was separated using the Oligotex mRNA Mini Kit (Qiagen Inc.). First-strand cDNA synthesis was performed using oligo(dT)₁₅primers. PCR amplification for E7 sequence was carried out with the following primer pair: 5′-GAAGATCTATCATGCATGGAGATACACC-3′ and 5-CTAGATCTTTATGGTTTCTGAGAACAGATGG-3′ that amplifythe HPV-16 sequence from nucleotides 139 to 420. A control PCR analysis consisting of amplification of the TF_IIB gene segment was included in all reactions. In addition, a control amplification using mRNA without prior RT was also included. PCR products were visualized by ethidium bromide staining on an ultraviolet light transilluminator.

4.7 Detection of viral integration by PCR/Southern blot analysis

Chromosomal integration of the AAV/E7/Neo genome was undertaken by vector-chromosome junction PCR amplification and Southern blot analysis, as previously described 15.

4.8 Generation and testing of E7-specific CTL

Nonadherent PBMC from the same healthy donor were washed and resuspended in AIM-V at 10×10⁶–20×10⁶ cells/well in six-well culture plates (Costar, Cambridge, MA) with rAAV- or GST-E7-pulsed DC (ratios from 20:1, responders:dendritic). The cultures were supplemented with recombinant human GM-CSF (800 U/ml) and recombinant human IL-2 (10 U/ml). At 7 days of the co-culture, the cells were used for cytotoxicity assays in a 6-h ⁵¹Cr release assay, as previously described 12.

4.9 Analysis for intracellular cytokines and protein

This protocol is adapted from that described by Pala et al. 34. The mixed T cell population was tested at 2 weeks post-priming, including 7 days of resting, after activationby PMA and ionomycin. Briefly, T cells (7.5×10⁵/ml) were incubated at 37^oC for 6 h in AIM-V 5% autologous plasma plus 50 ng/ml PMA and 500 ng/ml ionomycin. Brefeldin A, 10 ng/ml, was added for the final 3 h of incubation. Controls (nonactivated cultures) were incubated in the presence of Brefeldin A only. The cells were harvested, washed and fixed with 2% paraformaldehyde in phosphate buffered saline (PBS) for 20 min at room temperature. The cells were washed and permeabilized with PBS/1% bovine serum albumin (BSA)/0.5% saponin (S-7900, Sigma) for 10 min at room temperature. Activated and control cells were stained with FITC-anti-IFN-γ, and PE-anti-IL-4 and analyzed by flow cytometry. For intracellular E6 protein analysis, AAV/E6-E7/Neo-infected cells were stained with anti-HPV-16/18 E6 (Chemicon Inc., Temecula, CA; Cat MAB874) plus FITC-anti-mouse-Ig (Becton Dickinson Inc., cat no. 554001) and analyzed by flow cytometry.

4.10 Cell surface marker analysis of T cells and DC by fluorescent antibody cell sorting (FACS)

For the analysis of T cells at day 12 of the experiment the primed T cell populations were analyzed for surface markers. Monoclonal antibodies (mAb) recognizing the following antigens were used: anti-CD4, anti-CD8, and anti-CD56 (Pharmingen, San Diego, CA). Control irrelevant isotype-matched FITC- or PE-conjugated mAb were obtained from Becton Dickinson. These cells were >95% viableas assessed by trypan blue exclusion. Cell suspensions were counted and distributed into 12×75 mm tubes. Mouse mAb were diluted in cold assay buffer, and the final pellet was resuspended in a 500-μl volume. Tubes were incubated for 30 min and washed twice with assay buffer, and the final cell pellet was resuspended in 500 μl of assay buffer for subsequent analysis. Cells were analyzed with a fluorescence activated cell sorter (FACS; Becton Dickinson) with a 15-mW argon laser having an excitation of 488 nm. Fluorescent signals were gated on the basis of cell dimension (i.e. forward and right-angle light scattering typical of PBL activated). Gated signals (5,000–10,000) were detected at 585 BP filter and analyzed with Cell Quest software (Becton Dickinson).

For the analysis of DC, a panel of mAb recognizing the following antigens was used: anti-CD40 (Immunotech, Marseille, France); anti-CD14, anti-DR, anti-CD80 (Becton Dickinson), anti-CD86 (Pharmingen, San Diego, CA), and anti-CD83 (Coulter, Miami, FL). Briefly, nonadherent cells were harvested by washing the plates with PBS (pH 7.2, GibcoBRL). Adherent cells were recovered by incubatingthe plates at room temperature for 15–20 min in the presence of Ca²⁺- and Mg²⁺-free PBS, followed by gentle scraping. These cells were >95% viable as assessed by trypan blue exclusion. Cell suspensions were counted and distributed into 12×75 mm tubes. Mouse mAb antibodies were diluted in cold assay buffer (PBS, pH 7.2, supplemented with 0.1% fetal bovine serum) andadded in a 50-μl volume. For direct fluorescence, tubes were incubated for 30 min and washed twice with assay buffer, and the final cell pellet was resuspended in 500 μl assay buffer for subsequent analysis.