Cross-priming of cyclin B1, MUC-1 and survivin-specific CD8+T cells by dendritic cells loaded with killed allogeneic breast cancer cells
© Saito et al.; licensee BioMed Central Ltd. 2006
Received: 26 May 2006
Accepted: 27 November 2006
Published: 27 November 2006
The ability of dendritic cells (DCs) to take up whole tumor cells and process their antigens for presentation to T cells ('cross-priming') is an important mechanism for induction of tumor specific immunity.
In vitro generated DCs were loaded with killed allogeneic breast cancer cells and offered to autologous naïve CD8+ T cells in 2-week and/or 3-week cultures. CD8+ T cell differentiation was measured by their capacity to secrete effector cytokines (interferon-γ) and kill breast cancer cells. Specificity was measured using peptides derived from defined breast cancer antigens.
We found that DCs loaded with killed breast cancer cells can prime naïve CD8+ T cells to differentiate into effector cytotoxic T lymphocytes (CTLs). Importantly, these CTLs primed by DCs loaded with killed HLA-A*0201- breast cancer cells can kill HLA-A*0201+ breast cancer cells. Among the tumor specific CTLs, we found that CTLs specific for HLA-A2 restricted peptides derived from three well known shared breast tumor antigens, namely cyclin B1, MUC-1 and survivin.
This ability of DCs loaded with killed allogeneic breast cancer cells to elicit multiantigen specific immunity supports their use as vaccines in patients with breast cancer.
Despite declining mortality rates, breast cancer ranks second among cancer related deaths in women. In the USA alone, it is estimated that more than 200,000 new cases of breast cancer will be diagnosed yearly, with that about 40,000 patients will die from breast cancer . Therefore, there is a need to develop novel therapeutic approaches to improve survival rates among patients with breast cancer.
Evidence is accumulating that naturally occurring immunity is present in patients with breast cancer against tumor associated antigens such as HER-2/neu [2, 3] and cdr2 , as well as the antigens investigated here, namely MUC-1 [5, 6], cyclin B1  and survivin . Several clinical studies have now demonstrated that immunity against tumor antigens can also be boosted or elicited in cancer patients by vaccination, most recently through the use of tumor antigen loaded dendritic cells (DCs) [9–13]. For example, in a recently reported trial , two out of ten patients with breast/ovarian cancer vaccinated with HER-2/neu or MUC-1 peptide-pulsed monocyte-derived DCs generated cytotoxic T lymphocytes (CTLs) that were able to kill HLA-A*0201 cell lines expressing these antigens. The optimal source of tumor antigens for loading of ex vivo generated DCs is yet to be determined . In particular, strategies that allow tumor antigen presentation across HLA haplotypes are needed, and several have been undergoing investigation. These include loading DCs with recombinant proteins, killed tumor cells [4, 16–18], tumor RNA [19, 20] and viral vectors that encode tumor antigens , and fusing DCs with tumor cells . In prostate cancer  and in melanoma , we previously showed that DCs loaded with killed allogeneic tumor cells cross-prime naïve CD8+ T cells to differentiate into CTLs specific for shared tumor antigens.
In breast cancer, we previously demonstrated that DCs loaded with killed breast cancer cells can induce CTLs that can kill those breast cancer cells . However, we were unable to determine the antigen specificity of these CTLs. In an extension of that work, here we demonstrate that DCs loaded with killed allogeneic breast cancer cells can prime naïve CD8+ T cells to differentiate into tumor antigen specific CTLs by confirming their specificity for three known breast cancer antigens: cyclin B1, MUC-1, and survivin.
Materials and methods
Media and reagents
Complete RPMI-1640 was supplemented with 10% heat-inactivated fetal calf serum (Hyclone, Logan, UT, USA) for DCs and 10% human serum AB (Gem cell) for T cell cultures. Cytokines used included granulocyte-macrophage colony-stimulating factor (100 ng/ml; Immunex, Amgen Thousand Oaks, CA, USA), IL-4 (25 ng/ml; R&D Systems, Minneapolis, MN, USA), soluble CD40 ligand (200 ng/ml; R&D Systems), IL-2 (10 IU/ml; R&D Systems), and IL-7 (10 IU/ml; R&D Systems). Betulinic acid (Sigma-Aldrich, St. Louis, MO, USA) was used at 10 mg/ml. Cyclin B1 peptide CB9 (sequence AKYLMELTM) was synthesized at the University of Pittsburgh Cancer Institute Peptide Synthesis facility. MUC-1 peptides D6 (sequence LLLTVLTVV) and PSA1 (sequence FLTPKKLQCV) were purchased from Bio-Synthesis (TX, Lewisville, USA). The surviving peptide library was synthesized at the Rockefeller University.
Breast cancer cell lines T47D, Hs578T, MCF-7, natural killer cell target K562 and HLA-A*0201+ T2 cells, which are targets for CTLs, were obtained from the American Type Culture Collection (Manassas, VA, USA). The Me275 melanoma cell line was a kind gift from Drs J-C Cerottini and D Rimoldi. All cell lines were maintained in complete RPMI1640 (Gibco, Invitrogen, Carlsbad, California, USA). For loading of DCs, T47D and Hs578T cells were killed by incubation with betulinic acid (Sigma-Aldrich), as described previously . MCF-7 cells were killed by gamma irradiation (80 Gy) and 24 hours of exposure to tumor necrosis factor-α (100 ng/ml; R&D Systems).
Generation of cytotoxic T lymphocytes
Naïve HLA-A*0201+ CD8+ T cells (autologous to the DCs) were depleted of other cells using anti-CD4, anti-CD14, anti-CD16, anti-CD19, anti-CD56, and anti-glycophorin A microbeads (Miltenyi, Auburn, CA, USA) and sorted based on CD8+CD45RA+CCR7+CD45RO- phenotype. DCs were generated from the adherent fraction of peripheral blood mononuclear cells by culturing for 6 days in complete RPMI1640 supplemented with granulocyte-macrophage colony-stimulating factor and IL-4, loaded with killed tumor cells, sorted, and used as stimulators of autologous purified naïve CD8+ T cells at a 1:10 ratio. Soluble CD40 ligand was added to induce DC maturation, and IL-7 (10 IU/ml in weeks 1, 2, and 3) and IL-2 (10 IU/ml in weeks 2 and 3) for T cell expansion. T cells were restimulated weekly with tumor cell loaded DCs.
51Cr cytotoxicity assay
For use as targets, T2 cells were pulsed overnight with 10 μg/ml of various peptides, labeled with 51Cr (NEN Life Science Products, Boston, Massachusetts, USA) and co-cultured with CTLs for 4 hours. Specific lysis was calculated using the following formula (where cpm is the counts/minute): % release = 100 × (cpm experiment – cpm spontaneous release)/(cpm maximum release – cpm spontaneous release). For the mAb blocking assays, anti-HLA-A, HLA-B and HLA-C antibody (W6/32; DAKO, Carpenteria, CA, USA; 5 μg/ml) or an irrelevant (control) antibody was added to the target cells 30 minutes before the addition of CTLs and left throughout the culture period.
Tumor inhibition assay
Target tumor cell lines were suspended at a concentration of 5 × 104/ml with RPMI1640 medium containing 10% AB serum and the CTLs at 106/ml. Targets cells and CTLs were co-incubated in 96-U bottom plates (Costar, Corning, Sigma-Aldrich) for 24 hours. The numbers of live and dead tumor cells were determined using trypan blue exclusion.
Intracellular cytokine staining
Primed and boosted T cells were cultured for 6 hours with DCs loaded with 15-mer peptides representing survivin peptide library. At 2 hours of stimulation, Golgi-stop™ (BD Pharmingen, San Diego, CA, USA) was added to the culture. At 6 hours of stimulation, cells were harvested and first stained with CD3-PerCP and CD8-APC mAbs, fixed and permeabilized with BD Cytofix/Cytoperm™ solution (BD), and then stained with anti-IFN-γ PE (BD Pharmingen).
T cell restimulation assay
CD8+ T cells that had undergone two stimulations with DCs loaded with killed breast cancer cells were co-incubated with autologous DCs pulsed with the survivin peptide library at a 10:1 ratio. The T cells were analyzed after an additional 5 to 7 days of culture by intracellular cytokine staining, as described above.
Nonparametric Kruskal-Wallis analysis of variance was used as indicated to assess the killing of three different target cells. P < 0.05 was considered statistically significant.
Development of breast cancer specific CD8+T cells through cross-priming
Specificity of breast cancer cross-primed cytotoxic T lymphocytes for defined shared tumor antigens
The desired objective of loading DCs with killed breast cancer cells was to prime a polyclonal population of antigen specific CTLs. To determine whether this was indeed the case, we tested the primed CTL cultures for the presence of T cells specific for three well known tumor antigens expressed in breast cancers, namely cyclin B1, MUC-1 and survivin.
Cyclin B1-specific cytotoxic T lymphocytes
MUC-1 specific cytotoxic T lymphocytes
Survivin-specific T cells
Using different target antigens and different assays to measure CTL function, we demonstrated that DCs loaded with killed allogeneic breast cancer cells cross-prime naïve CD8+ T cells to differentiate into breast cancer specific effector CTLs. The elicited CTLs demonstrate specific effector function, as measured by their capacity to kill breast cancer cells used for priming, to kill T2 cells pulsed with defined peptides derived from breast cancer antigens, and to secrete IFN-γ upon peptide exposure. Among the primed CTLs, we demonstrate at least three specificities against the known shared breast tumor antigens cyclin B1, MUC-1, and survivin.
Interestingly, we observed differences in the priming of CD8+ T cells with distinct specificities that were consistent with the unique nature of each antigen. Thus, T cells specific for cyclin B1 could be detected after priming and two rounds of boosting with DCs loaded with killed breast cancer cells. However, this strategy did not permit detection of MUC-1 specific T cells, which required an additional boost with MUC-1 peptide pulsed DCs. We ascribe this to the differences in efficiency of presentation of different antigens by loaded DCs and/or differences in the frequency of precursor T cells with these specificities. Indeed, cyclin B1 represents a protein antigen whose function as a tumor antigen is associated with increased cytoplasmic accumulation followed by degradation through ubiquitination . This might make it more accessible to the DC antigen processing/presentation machinery than the transmembrane MUC-1 glycoprotein. Previous studies by some of us [25–27] demonstrated that MUC-1 and HER-2/neu are retained in DC early endosomes, leading to inefficient processing and cross-presentation to T cells. These observations, together with our earlier study and the lack of specificity to some other defined breast cancer antigens, including HER-2/neu , suggest that efficient cross-presentation of individual tumor antigens by DCs loaded with killed breast cancer cells will depend on the nature of these antigens or the form in which they are taken up by DCs. For example, although we found that DCs loaded with whole tumor cells are not efficient in cross-presenting MUC-1, a recent study  demonstrated that loading DCs with breast cancer cell lysates permits more efficient priming of MUC-1 specific CD8+ T cells. It is difficult to determine whether the difference in the outcomes between that study and ours is due to a real difference between loading DCs with whole tumor cells and loading them with tumor cell lysate, or to a difference in how the MUC-1 specific T cells were detected. CD8+ T cells in that report were measured based on tetramer binding but in our study measurement was based on a functional assay, specifically the activity of CTLs against target cells expressing MUC-1 peptide.
Thus, our data offer experimental proof that using DCs loaded with killed allogeneic breast cancer cells for antigen presentation leads to priming of T cells to multiple tumor antigens, some that are already known and could be tested here, and potentially many more unknown but nevertheless important shared tumor antigens. The advantage of this approach is the ability to generate, with one vaccine, T cells specific for many tumor antigens and in many patients, regardless of HLA type. Although it is conceivable that part of the generated response might be due to HLA mismatch, breast cancer tumor antigen specific immune responses were also generated. Finally, we previously demonstrated the validity of this approach in patients with stage IV melanoma . In that study patients were vaccinated with DCs loaded with killed allogeneic melanoma cells. We that melanoma specific immune responses were induced and, perhaps more importantly, durable (>33 months) objective clinical responses were identified in 10% of patients in whom conventional therapy failed. Thus, this vaccination strategy might now be tested in phase I studies in patients with metastatic breast cancer.
This ability of DCs loaded with killed allogeneic breast cancer cells to elicit multiantigen specific immunity supports their use as vaccines in patients with breast cancer.
cytotoxic T lymphocyte
We thank Dr Joseph Fay, Bi-Jue Chang, Nathalie Piqueras, Doris Wood, and Susan Hicks for help with recruitment and follow up of healthy volunteers. We thank Lynnette Walters at BIIR Cell and Tissue Procurement Core; Susan Burkeholder, Jennifer Finholt-Perry and Fabienne Kerneis at BIIR GMP Lab; and Elizabeth T Kraus and Sebastien Coquery at BIIR Flow Cytometry Core for technical help; and Cindy Samuelsen and Nicolas Taquet for invaluable help. We thank Dr Michael Ramsay for continuous support.
Supported by NIH grants CA89440 (AKP), PO-1 CA84512 (AKP and JB), CA78846 (JB), CA56103 (OJF) and CA090440 (OJF); and FWFAustria (Schroedinger Stipendium, J2255-B08: PD). JB holds Caruth Chair for Transplant Immunology Research. AKP holds Ramsay Chair for Cancer Immunology Research.
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