Antigen-targeted immunotherapy is an emerging treatment for breast cancer. However, useful breast cancer antigens are only found in a subset of cancer patients. BA46, also known as lactadherin, is a membrane-associated glycoprotein that is expressed in most breast cancer cells but not in general hematopoietic cell populations. Moreover, it is much more difficult to generate CTLs against self-antigens. We wished to determine if the use of recombinant adeno-associated virus (rAAV) type 2 vectors for gene-loading of dendritic cells (DCs) could generate rapid, effective cytotoxic T lymphocytes (CTLs) against BA46. We were able to demonstrate that AAV/BA46/Neo-loading of DCs resulted in: (1) BA46 expression in DCs, (2) chromosomal integration of the AAV/BA46/Neo vector within DCs, (3) strong, rapid BA46-specific, MHC class I-restricted CTLs in only 1 week, (4) T-cell populations with significant interferon-γ (IFN-γ) expression but low IL-4 expression, (5) high CD80 and CD86 expression in DCs, and (6) high CD8:CD4 and CD8:CD56 T cell ratios. These data suggest that rAAV-loading of DCs may be useful for immunotherapeutic protocols against self-antigens in addition to viral antigens and that the BA46 antigen is potentially appropriate for cell-mediated immunotherapeutic protocols addressing ductal breast cancer.
Antigen-targeted immunotherapy has joined the arsenal of chemotherapy and radiation therapy for the treatment of breast cancer. The search for useful breast cancer antigens has met with some success, identifying Her-2/neu and folate-binding protein as possible targets.1, 2, 3, 4, 5 However, these antigens are only found in a subset of cancer patients. BA46, also known as lactadherin, is a membrane-associated glycoprotein that is expressed in most breast cancer cells. Larocca et al6 demonstrated its expression in seven of seven breast cell lines tested. BA46 is also a component of milk, commonly referred to as milk fat globular membrane.6, 7, 8, 9 We wished to determine if BA46 would be an appropriate anti-breast cancer antigen using recombinant adeno-associated virus (AAV)-based gene loading of dendritic cells (DCs).
DCs are potent, professional antigen-presenting cells that are able to initiate a primary immune response to antigens by naive T cells.10 Protocols for generating DCs in vitro from peripheral blood have been developed, and these new technologies permit the in vitro manipulation of DCs for clinical and laboratory immunotherapeutic studies.11, 12 These protocols include loading or incubating DCs with tumor lysates, tumor fragments, antigen peptides, specific tumor proteins, or with antigen genes by way of retrovirus or adenovirus vectors.13, 14, 15, 16, 17, 18, 19, 20 AAV type 2 has been shown in various studies to be an effective gene delivery vector for both immortalized tissue culture cells as well as primary hematopoietic cells.21, 22, 23, 24 We have shown that AAV type 2 can be used to transduce both cytokine and antigen genes into primary human monocytes (Mo) and derived DCs with high efficiency (70–90+%).25, 26, 27 The use of AAV-based DC loading of the human papillomavirus E6 and E7 antigen genes resulted in significant antigen-specific, MHC Class I-restricted cytotoxic T lymphocyte (CTL) activity with one stimulation (one DC addition) after 1 week coincubation.26, 27
However, these previous studies generated CTLs against non-self-viral antigens. Self-antigen-recognizing T-cell precursors are much less numerous than antiviral antigen precursors, thus making it more difficult to generate CTLs against self-antigens.28, 29 In this study, we were able to demonstrate that the rAAV-DC-loading technique could be used to rapidly generate CTLs against the self-antigen BA46. The uniqueness of BA46 as a breast cancer antigen was also addressed, and BA46 was shown not to be highly expressed in general hematopoietic cell populations. These data suggest that rAAV/antigen-DC loading may be a useful technique for immunotherapeutic protocols against self- or weak antigens and that BA46 might be an appropriate target for anti-breast cancer antigen therapy.
Materials and methods
Generation of BA46 cDNA
BA46 cDNA was generated by RT-PCR amplification from Hs 578T breast cancer cells (ATCC HTB-126; Manassas, VA). Total RNA was isolated (Trizol reagent, Invitrogen, Carlsbad, CA) and treated with 5 U/mg of RNase-free DNase I (Promega, Madison, WI) at 37°C for 2 hours. The mRNA was separated using the Oligotex mRNA Mini Kit (QIAGEN, Valencia, CA). First-strand RT-based cDNA synthesis was performed using oligo(dT)15 primers. PCR amplification for the BA46 sequence was carried out using the following primer pair, which amplifies the sequence from nucleotides −7 to 1167.8
An RT-PCR amplification of the TFIIB mRNA segment was also included as a control.26, 27 To ensure that DNA was not contributing to the results, a direct PCR amplification, using total mRNA without reverse transcription, was also performed. The BA46 cDNA product was then gel-purified using the GENECLEAN III KIT (Bio 101, CA), and the product was used in the construction of the AAV/BA46/Neo and AAV/BA46/zeocin plasmids, as described below.
Analysis of BA46 mRNA expression
BA46 mRNA expression was detected in transduced DCs, PBMCs, and K562 cells using RT-PCR amplification along with a cellular mRNA control. Total polyA-selected mRNA was isolated from infected and mock-infected cells and cDNAs for BA46 and TFIIB generated as above. PCR products were visualized by ethidium bromide staining on an ultraviolet light transilluminator. The resulting BA46 RT-PCR products were Southern blotted and probed with 32P-labeled BA46 sequences to observe expression in the most sensitive and specific manner.
Construction of AAV/BA46/Neo and AAV/BA46/zeocin virus plasmids
The AAV/BA46/Neo and AAV/BA46/zeocin genomes were constructed using a strategy similar to that previously described for the AAV/GM-CSF/Neo viral genome.25 Briefly, the BA46 open reading frame cDNA was ligated into dl6-95, a basic gutted AAV vector containing the SV40 early promoter neomycin (Neo)-resistant gene transcription cassette. The SV40 early promotor-zeocin transcription cassette was derived from plasmid pSecTag2 (Invitrogen, Carlsbad, CA) via PCR and was used to replace the SV40 early promoter-Neo DNA. At this stage, the BA46 cDNA was sequenced and determined to be identical to the published sequence.8
Generation of AAV/BA46/Neo and AAV/BA46/zeocin virus stocks
Adenovirus-free AAV/BA46/Neo and AAV/BA46/zeocin virus stocks were generated using the complementor plasmid pSH3, as described previously using 293 cells.30, 31 The Neo-resistant gene or zeocin-resistant gene were included, so we could generate rAAV producer cell lines with high-titer virus stocks.25, 26, 27 AAV/BA46/zeocin virus was used to generate autologous target cells for CTL analysis that were BA46-positive/Neo-negative. To generate purified rAAV virus, the technique described by Auricchio et al32 was used. Briefly, after DNase I-treatment (10 U/ml for 1 hour), the virus solution was incubated with 0.5% deoxycholic acid (Sigma, St Louis, MO) for 30 minutes at 37°C then purified on a heparin–agarose column (Sigma) using phosphate-buffered saline (PBS, pH 7.0) as an eluent. The eluate was concentrated to approximately 6 ml. The virus stocks were titered by extracting viral DNA and carrying out a comparative dot blot hybridization, as described previously,25, 26, 27 yielding titers of approximately 1 × 1010 encapsidated genomes per ml (eg/ml).
Generation and antigen loading of DC
The study protocol conformed to the ethical guidelines of the 1975 Declaration of Helsinki, as reflected in a priori approval by our Human Research Advisory Committee. Peripheral blood was derived from six healthy donors. Ficoll gradient-purified PBMCs were inoculated into six-well culture plates for 2 hours at 37°C and 5% CO2, and the adherent cells were selected following three gentle washes. Immediately after the removal of the nonadherent cells, the adherent Mo were infected with 1 × 108 eg of AAV/BA46/Neo virus. After 4 hours of incubation, the medium/virus solution was removed, the cells washed, and fresh AIM-V medium (Invitrogen Life Technologies, Gaithersburg, MD) added. The Mo/DC precursors were infected immediately at the time of removal of the nonadherent cells with AAV/BA46/Neo virus stock on day 0. GM-CSF (Immunex, Seattle, WA) at a final concentration of 800 IU/ml was included in the medium throughout the culture. To induce the differentiation of Mo into DCs, human IL-4 (R&D SYSTEMS, Minneapolis, MN) at 1000 IU/ml was added at day 3.
Cell surface marker analysis of DCs by flow cytometry
For the characterization of DCs, a panel of mAb recognizing the following antigens was used: anti-CD14, anti-CD40, anti-HLA-DR, anti-CD80 (Caltag Laboratories, Burlingame, CA), and anti-CD86 (BD Pharmingen, San Diego, CA). Control irrelevant isotype-matched FITC- or PE-conjugated mAb were obtained from Caltag Laboratories. Briefly, non-adherent cells were harvested by washing the plates with PBS, pH 7.2. Cell suspensions were counted and distributed into 12 × 75 mm tubes. Mouse monoclonal antibodies were diluted in cold assay buffer (PBS, pH 7.2, supplemented with 1% FBS) and added in a 50-ml volume. For direct fluorescence, tubes were incubated for 30 minutes, followed by two washes with assay buffer, and the final cell pellet was resuspended in 500-ml assay buffer for subsequent analysis.
Detection of viral integration in DCs by PCR/Southern blot analysis
The primary breast cancer cell line Hs578T (HLA-A1 haplotype) was used as one BA46-positive target. The HLA haplotypes of all donors were compatible with this cell line. The autologous donor EBV-transformed lymphoblastoid cell lines (LCLs) were generated according to the routine method and were derived from the six healthy donors. The LCLs were infected with AAV/BA46/zeocin virus (no Neo) at a multiplicity of infection of 10 eg/cell and then cultured in AIM-V medium containing zeocin (20 μg/ml) for more than 15 days. Before the 51Cr-release assay, RT-PCR was performed to check for expression of BA46 mRNA (data not shown).
Generation and testing of BA46-specific CTLs
On day 5, non-adherent PBMCs from the same healthy donors were washed and added in AIM-V at 10–20 × 106 cells/well to AAV/BA46/Neo-loaded or mock-treated DCs (ratio of 20:1, responders:dendritic) in six-well culture plates. The cultures were supplemented with recombinant human GM-CSF (800 U/ml), recombinant human IL-2 (10 U/ml, R&D SYSTEMS, Minneapolis, MN), and IL-7 (20 ng/ml, R&D SYSTEMS, Minneapolis, MN). Where indicated, target cells were preincubated with anti-Class I (monomorphic-A, -B, -C) blocking antibody at 1/1000 (Biomeda, Foster City, CA). After 7 days of coculture (day 12 of Fig 3c), the cells were used for cytotoxicity assays in a 6-hour 51Cr-release assay, as previously described.26, 27, 33
Analysis of T cells for intracellular cytokines
This protocol is adapted from that described by Pala et al.34 The mixed T-cell population was tested at 7 days postpriming. Briefly, the cells were harvested, washed, and fixed with 2% paraformaldehyde in PBS for 20 minutes at room temperature. The cells were washed and permeabilized with PBS/1% BSA/0.5% saponin (Sigma, St Louis, MO) for 10 minutes at room temperature. Activated and control cells were stained with FITC-anti-IFN-γ and PE-anti-IL-4 and analyzed by flow cytometry.
Cell surface marker analysis of stimulated T cells
The primed T-cell populations were analyzed for surface markers on day 12. A panel of mAbs recognizing the following antigens was used: anti-CD4, anti-CD8, and anti-CD56 (Caltag Laboratories, Burlingame, CA). Control irrelevant isotype-matched FITC- or PE-conjugated mAbs were obtained from Caltag Laboratories. The flow cytometric analysis was the same as described above.
Analysis of BA46 expression in general PBMC population
Although BA46 appears to be expressed in most breast carcinomas, one group has observed BA46 expression in K562 and other hematopoietic cells.36 Such expression might limit the usefulness of BA46 as an anti-breast cancer antigen. To address the novelty and specificity of BA46 as a ductal/mammary epithelial antigen, we observed the expression of BA46 in K562 cells as well as in PBMCs isolated from three different donors of both sexes. The analysis was carried out by RT-PCR and Southern blot analysis. This combined technique is very specific and very sensitive. As shown in Figure 1, while expression was seen in a representative ductal breast cancer cell line, no expression was observed in the PBMC samples nor in the K562 cells. These data, coupled with the lack of recognition (killing) of unaltered peripheral blood lymphocytes (PBLs) and K562 cells (to be shown in Fig 7a and b), strongly suggest that BA46 is not significantly expressed in hematopoietic cells.
AAV/BA46/Neo-transduced DCs express BA46
Figure 2 shows that the BA46 cDNA clone generated as an RT-PCR product from the Hs578T cell line did result in a product of the expected size. This cDNA was then ligated into the basic AAV/Neo vector dl6-95/Neo to give AAV/BA46/Neo. Figure 3a shows a structural map of the AAV/BA46/Neo vector, which was used in this study. In these vectors, the BA46 gene was expressed from the AAV p5 promoter, known to be active in DCs.25, 26, 27 Figure 3b shows the titer of two AAV/BA46/Neo virus stocks. A stock of AAV/BA46/zeocin virus was also generated and titered (data not shown).
The transduction of the Mo/DC population was first observed by measuring polyadenylated RNA expression of the BA46 transgene. A schematic diagram of the experimental protocol is shown in Figure 3c. At day 5, mRNA was analyzed for BA46 expression by RT-PCR. A cellular gene, TFIIB, was used as a control. As shown in Figure 4, BA46 expression was only detected in the AAV/BA46/Neo-infected DCs, not in mock infected cells.
AAV/BA46/Neo infection results in proviral chromosomal integration
We next observed chromosomal integration of the AAV/BA46/Neo vector in DCs. 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 for proviral integration was carried out by PCR amplification of vector–chromosome junctions using primers complementary to the SV40 promoter within the vector and Alu I repetitive chromosomal elements.25, 26, 27 AAV-cell junction products were analyzed by PCR amplification and Southern blot analysis, probing for the Neo gene sequences. Multiple vector–chromosomal junction products were observed in the AAV/BA46/Neo-infected DCs but not in mock-infected DCs, as shown in Figure 5. These data indicate that at least a subset of the viral genomes is able to integrate into the DC population chromosomally.
Characterization of DCs under various treatments
We used flow cytometric analysis to determine the surface marker phenotype of mock-loaded, wild-type AAV-loaded, and AAV/BA46/Neo-loaded DC populations. The results, shown in Figure 6, demonstrate that the DCs generated from all three techniques share common DC markers and low CD14 levels. However, wild-type AAV-loaded DCs and AAV/BA46/Neo-loaded DCs expressed significantly higher levels of CD80. AAV/BA46/Neo-loaded DCs also expressed significantly higher levels of CD86 and CD40.
AAV/BA46/Neo-loaded DC-stimulated BA46-specific CTLs are effective tumor cell killers
The ability of the AAV/BA46/Neo virus antigen loading technique to generate CTLs with only one DC stimulation was analyzed. DCs were loaded and CTLs generated as experimentally structured in Figure 3c. Figure 7a shows a representative of five experiments and demonstrates that the T-cell population derived from coculture with AAV/BA46/Neo-loaded DCs were able to lyse MHC Class I-matched cells of the BA46-positive Hs578T breast cancer cell line (gray bars). This indicates the ability of AAV/BA46/Neo-loaded DCs to prime and propagate BA46-specific CTLs.
Furthermore, when anti-BA46 CTL activity was stimulated in a dose-dependent manner with the amount of virus used to load the DCs, little activity was seen against the MHC Class I-matched target cell line K562 (cross hatched bar). These cells are often used as a target to observe natural killer (NK) target cell killing. The addition of anti-MHC class I-antibodies blocked the killing by the AAV/BA46/Neo-stimulated CTLs. None of the two controls, mock- or wild-type AAV-loaded DCs, was able to prime and propagate anti-BA46 CTLs, further supporting the antigen-specificity of the CTL killing. Finally, Figure 7a shows high CTL killing of the BA46-positive breast cancer cells but low killing of K562 cells. K562 cells should be targeted if they express BA46 protein. Taken together, these data strongly suggest that the killing of target cells was BA46-specific, Class I-restricted, and that tumor cell lysis was not mediated through NK cells.
The stimulation of anti-BA46 CTLs was then observed in a fully autologous system. The CTLs were generated as in Figure 5a, with the exception of using the same donor for all three cell types used in the experiment (DCs, CTLs, and targets). The targets were autologous AAV/BA46/zeocin virus-infected LCLs. The results, shown in Figure 7b, were similar to those shown in Figure 7a (black bars), including evidence of the ability of a monoclonal antibody directed at the monomorphic MHC Class I-molecules to block killing. Furthermore, this experiment also shows that unaltered LCL targets, without BA46 antigen introduction (empty bar, far left lane), were not targeted for killing by the AAV/BA46/Neo-stimulated CTLs. The mock-loaded DC-stimulated CTLs had very little killing activity upon the BA46-positive target cells. Along with the experiments represented in Figure 7a, these data specifically and effectively demonstrates the high antigen specificity of these anti-BA46 CTL. These data are also consistent with Figure 1, strongly suggesting a lack of significant BA46 expression in general hematopoietic cells.
Cytokine profile and characterization of AAV/BA46/Neo-loaded DC-stimulated CTLs
To determine the cytokine profile of the T cells generated from coculture with AAV/BA46/Neo-transduced DCs or mock-loaded DCs, we carried out intracellular staining of these T cells for IFN-γ and IL-4 (day 12). Figure 8 demonstrates that the mock-generated T cells expressed IFN-γ and IL-4 at approximately equal frequency. In contrast, the AAV/BA46/Neo-derived T cells expressed IFN-γ at a modestly higher frequency but expressed very little IL-4, suggesting that these T cells are more consistent with a Th1 response phenotype.
The T cells resulting from the mock- and AAV/BA46/Neo-loading techniques (day 12) were further characterized to observe if significant differences in surface markers were discernable. The results of flow cytometric analysis, shown in Figure 9a, demonstrate that the T cells treated with AAV/BA46/Neo had a much higher CD8:CD4 ratio than the mock-derived T cells. This is consistent with a strong MHC Class I-restricted CTL response. A similar analysis for CD8 and CD56 surface markers, to determined the potential involvement of NK cells in target cell killing, shows that the CD8:CD56 ratio was much higher with the AAV/BA46/Neo treatment than with the mock treatment, as shown in Figure 9b. These data are consistent with the data shown in Figure 7a, in which T cells derived from AAV/BA46/Neo were unable to kill K562 cells (BA46-negative).
In this study, we demonstrated that the rAAV/BA46/Neo virus is able to effectively load DCs, resulting in functionally active BA46 epitope-presenting DCs. These rAAV-transduced DCs were able to prime and propagate BA46 antigen-specific CTLs in an efficient manner. We have hypothesized that antigen gene loading of DCs by AAV may be more efficient than protein loading of DCs for several reasons.
First, due to protein degradation and MHC molecule cycling, protein loading of DCs may be an inefficient way to deliver an antigen. In contrast, gene transfer leads to a continuous production of the antigenic protein and may provide the opportunity for repeated rounds of presentation and CTL stimulation. Second, viral entry into cells is usually more efficient than proteins delivered via lipofection (92% vs 29%, respectively).26, 27 Third, epitopes of proteins produced in bacteria may be less immunoreactive as bacteria lack many of the post-translational modifications seen in mammals. Finally, when compared to the use of synthetic peptides for loading DCs, viral delivery of the full gene, encoding the full protein, will result in multiple epitopes being expressed by the DCs.
Protocols for the generation of DCs by stimulating the differentiation of PBMCs usually involve treating adherent Mo with GM-CSF and IL-4. We have modified these protocols in order to promote AAV vector transduction in DC precursor monocytes. This altered protocol involves treating adherent Mo with GM-CSF alone for several days before the addition of IL-4 on day 3.25, 26, 27 This allows the monocytes to go through a brief period of limited cell division. This cell division is important for promoting a higher level of AAV transduction.37, 38
While the generation of significant anti-BA46 CTLs and IFN-γ activity resulting from AAV/antigen gene loading of DCs was not surprising, the ability to generate significant CTL activity against a self-antigen with only one stimulation was surprising. Most DC loading/priming protocols require additional stimulations. We believe it possible that the higher CD80 expression in the DCs loaded with AAV/BA46/Neo is noteworthy. Other studies of AAV-loading of DCs confirm CD80 upregulation.26, 39 CD80 may be a more independent costimulatory molecule than CD86,40 and this may explain the improved capabilities of the vector-transduced DCs.26, 27
A number of breast cancer antigens have been identified; however, most are expressed in only a limited subset of breast cancers. We have chosen to target the milk/breast cancer antigen BA46 because of its broad expression in breast carcinomas and minimal expression in other cell types.6, 7, 8, 9 There has been a recent report that BA46 is also expressed in some hematopoietic cells,36 which would limit its utility as an antibreast cancer antigen. However, our RT-PCR/Southern blot analysis of K563 and PBLs from multiple donors, including both females and males, failed to find even small levels of BA46 mRNA. We also observed little CTL activity against K562 cells or autologous PBLs without the introduction of the BA46 antigen into these targets. Thus, we believe that endogenous BA46 expression in hematopoietic cells must be quite low if at all.
Taken together, these data support the usefulness of BA46 as an antibreast cancer antigen and the utility of AAV/antigen-gene loading of DCs for the generation of CTLs against self-antigens. Although another group has recently successfully used AAVs for the purpose of antigen gene-loading DCs to generate CTLs against a self-antigen,41 the rapid generation of CTLs that we observed after only one stimulation represents a higher order of accomplishment. There has also been a recent report on the use of BA46-based peptides for the generation of anti-BA46 CTLs.35 In the future, it would be interesting to compare peptide-loading with AAV/BA46-loading of DCs for BA46, as we have done for the human papillomavirus E6 and E7 antigens, to determine which technique is superior.
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Drs Yong Liu and Maurizio Chiriva-Internati contributed equally to this work. This work was funded by a grant from the Arkansas Foundation for Breast Cancer Research.
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Cite this article
Liu, Y., Chiriva-Internati, M., You, C. et al. Use and specificity of breast cancer antigen/milk protein BA46 for generating anti-self-cytotoxic T lymphocytes by recombinant adeno-associated virus-based gene loading of dendritic cells. Cancer Gene Ther 12, 304–312 (2005) doi:10.1038/sj.cgt.7700785
- adeno-associated virus
- dendritic cell
- breast cancer
- cytotoxic T lymphocyte
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