Cancer Res Treat.  2011 Mar;43(1):56-66.

Alpha-Type 1 Polarized Dendritic Cells Loaded with Apoptotic Allogeneic Breast Cancer Cells Can Induce Potent Cytotoxic T Lymphocytes against Breast Cancer

• Park MH
• Yang DH
• Kim MH
• Jang JH
• Jang YY
• Lee YK
• Jin CJ
• Pham TN
• Thi TA
• Lim MS
• Lee HJ
• Hong CY
• Yoon JH
• Lee JJ

Abstract

PURPOSE
Various tumor antigens can be loaded onto dendritic cells (DCs) to induce a potent cytotoxic T lymphocyte (CTL) response in DC-based immunotherapy against breast cancer. However, in the clinical setting, obtaining a sufficient number of autologous tumor cells as a source of tumor antigens is a laborious process. We therefore investigated the feasibility of immunotherapy using breast-cancer-specific CTLs generated in vitro by use of alpha-type 1 polarized DCs (alpha DC1s) loaded with ultraviolet B-irradiated cells of the breast cancer cell line MCF-7.
MATERIALS AND METHODS
alphaDC1s were induced by loading allogeneic tumor antigen generated from the MCF-7 UVB-irradiated breast cancer cell line. Antigen-pulsed alphaDC1s were evaluated by morphological and functional assays, and the breast-cancer-specific CTL response was analyzed by cytotoxic assay.
RESULTS
The alphaDC1s significantly increased the expression of several molecules related to DC maturation without differences according to whether the alphaDC1s were loaded with tumor antigens. The alphaDC1s showed a high production of interleukin-12 both during maturation and after subsequent stimulation with CD40L, which was not significantly affected by loading with tumor antigens. Breast-cancer-specific CTLs against autologous breast cancer cells were successfully induced by alphaDC1s loaded with apoptotic MCF-7 cells.
CONCLUSION
Autologous DCs loaded with an allogeneic breast cancer cell line can generate potent breast-cancer-specific CTL responses. This may be a practical method for cellular immunotherapy in patients with breast cancer.

Keyword

Dendritic cells; Breast neoplasms; Allogeneic; Cytotoxic T lymphocytes; Immunotherapy

MeSH Terms

Antigens, Neoplasm
Breast
Breast Neoplasms
CD40 Ligand
Cell Line
Dendritic Cells
Humans
Immunotherapy
Interleukin-12
Lymphocytes
T-Lymphocytes, Cytotoxic
Antigens, Neoplasm
CD40 Ligand
Interleukin-12

Figure

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  Fig. 1 Schema of the generation of dendritic cells (DCs) in patients with breast cancer in this study. PBMCs, peripheral blood mononuclear cells; D, day; GM-CSF, granulocyte-macrophage colony-stimulating factor; IL, interleukin; TNF, tumor necrosis factor; IFN, interferon; poly-I : C, polyinosinic : polycytidylic acid; αDC1, alpha-type 1 polarized DCs; IMDM, Iscove's Modified Dulbecco's medium; FBS, fetal bovine serum; UVB, ultraviolet B.

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  Fig. 2 Phenotypic expression of dendritic cells (DCs). The expressions of several molecules (CD83, CD86 and CCR7) related to DC maturation were significantly higher in the alpha-type 1 polarized DCs (αDC1s) than in the immature DCs (iDCs). However, there were no differences in phenotypic expression between the αDC1s and the αDC1s loaded with apoptotic MCF-7 breast cancer cells. Data are from one representative experiment of four independent experiments.

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  Fig. 3 Efficacy of tumor antigen uptake by dendritic cells (DCs). DCs were loaded with PKH67-labeled UVB-irradiated MCF-7 breast cancer cells (a ratio of 2 : 1) 2 hr after the addition of maturation-inducing cytokines on day 6. Mature DCs were harvested on day 8 and identified by CD11c expression. (A) The tumor antigen uptake of the alpha-type 1 polarized DCs (αDC1s) was measured by the percent of double-positive cells using flow cytometry. (B) After labeling MCF-7 cells with PKH67-GL-Green and DCs with PKH26-GL-Red, cell morphology was observed by using a confocal microscope at an excitation wavelength of 350 nm for DAPI. Apoptotic MCF-7 cells (yellow) were confirmed in the αDC1s.

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  Fig. 4 Comparison of cytokine production by dendritic cells (DCs). Production of interleukin (IL)-12p40, IL-12p70, IL-10 and IL-6 during maturation of the alpha-type 1 polarized DCs (αDC1s) and the αDC1s loaded with apoptotic tumor cells and during re-stimulation with 40L-tranfected J558 cells for 24 hr after maturation. Results, expressed as mean (pg/mL)±standard deviation of triplicate cultures, are from one representative experiment of three. iDCs, immature DCs.

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  Fig. 5 Alpha-type 1 polarized DCs (αDC1s) induce naïve CD4+ T cells to secrete Th1 cytokines. Allogeneic naïve CD4+CD45RA+ T cells were primed with DCs for 5 days and then stimulated with rIL-2. On day 10, the cells were re-stimulated with Dynabeads CD3/CD28 T cell expander for 1 day. (A) Interleukin (IL)-4 and interferon (IFN)-γ levels measured in culture supernatants by enzyme-linked immunosorbent assay. (B) IL-4 and IFN-γ expression by intracellular staining at the single-cell level. The results are one representative experiment of three independent experiments and are expressed as the percent of cytokine-producing cells. iDCs, immature DCs.

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  Fig. 6 Comparison of allogeneic T cell stimulatory capacities of dendritic cells (DCs). In a carboxyfluorescein diacetate succinimidyl ester (CFSE)-based proliferation assay, CFSE-labeled allogeneic CD3+ T cells (20,000 cells/well) were stimulated for 5 days with graded doses of irradiated DCs. Flow cytometry was used to measure the percent of CFSE-proliferated cells. The stimulatory capacity of the alpha-type 1 polarized DCs (αDC1s) was significantly higher in a dose-dependent manner than that of the immature DCs (iDCs), and there was no effect on T cell proliferation by loading of apoptotic tumor cells onto αDC1s. Data are representative histograms of fluorescence-activated cell sorting analysis from one of three independent experiments.

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  Fig. 7 Ezyme-linked immunospot (ELISPOT) assay to measure the interferon (IFN)-γ release of breast-cancer-specific cytotoxic T lymphocytes (CTLs). Autologous CD3+ T cells were stimulated with dendritic cells (DCs) loaded with apoptotic MCF-7 cells two times every 10 days. On day 20, the CTLs were co-cultured with autologous target cells (MCF-7, HLA-A0201+) and irrelevant target cells (T-47D, HLA-A0201- breast cancer cells; PC3, prostate cancer cells; K562, natural killer cell-sensitive chronic myeloid leukemia cells). (A) Number of IFN-γ-secreting cells from CTLs stimulated with the MCF-7-loaded-alpha-type 1 polarized DCs (αDC1s) (A) and the MCF-7-unloaded-αDC1s (B). Anti-major histocompatibility complex (MHC) class I antibodies were used to confirm the MHC class I-restricted recognition of breast-cancer-specific CTLs. Breast cancer cells were isolated from tumor tissue of patients and the CTLs were co-cultured with the breast cancer cells as target cells (C). The ELISPOT data are the mean (±standard deviation) number of IFN-γ-producing cells of triplicate cultures in three independent experiments.

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