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. 2009 Aug;27(8):1734-40.
doi: 10.1002/stem.102.

Antigen-specific T-cell response from dendritic cell vaccination using cancer stem-like cell-associated antigens

Qijin Xu  1 , Gentao Liu, Xiangpeng Yuan, Minlin Xu, Hongqiang Wang, Jianfei Ji, Bindu Konda, Keith L Black, John S Yu
Affiliations

Antigen-specific T-cell response from dendritic cell vaccination using cancer stem-like cell-associated antigens

Qijin Xu et al. Stem Cells. 2009 Aug.

Abstract

Glioblastoma multiforme (GBM) is the most aggressive primary brain tumor, with current treatment remaining palliative. Immunotherapies harness the body's own immune system to target cancers and could overcome the limitations of conventional treatments. One active immunotherapy strategy uses dendritic cell (DC)-based vaccination to initiate T-cell-mediated antitumor immunity. It has been proposed that cancer stem-like cells (CSCs) may play a key role in cancer initiation, progression, and resistance to current treatments. However, whether using human CSC antigens may improve the antitumor effect of DC vaccination against human cancer is unclear. In this study, we explored the suitability of CSCs as sources of antigens for DC vaccination again human GBM, with the aim of achieving CSC-targeting and enhanced antitumor immunity. We found that CSCs express high levels of tumor-associated antigens as well as major histocompatibility complex molecules. Furthermore, DC vaccination using CSC antigens elicited antigen-specific T-cell responses against CSCs. DC vaccination-induced interferon-gamma production is positively correlated with the number of antigen-specific T cells generated. Finally, using a 9L CSC brain tumor model, we demonstrate that vaccination with DCs loaded with 9L CSCs, but not daughter cells or conventionally cultured 9L cells, induced cytotoxic T lymphocytes (CTLs) against CSCs, and prolonged survival in animals bearing 9L CSC tumors. Understanding how immunization with CSCs generates superior antitumor immunity may accelerate development of CSC-specific immunotherapies and cancer vaccines.

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Figures

Figure 1
Figure 1
Glioblastoma-derived spheres are enriched in cells expressing cancer stem-like cell markers. A. Glioblastoma-derived cancer stem-like cells express CD133, and Nestin. Primary cultured glioblastoma-derived spheres were processed for immunofluorescence staining using antibodies against human CD133 and Nestin. DAPI staining was used to reveal cell nuclei. B. Glioblastoma-derived spheres are enriched in cells expressing both CD133 and CD15. Glioblastoma-derived spheres were processed into single cell suspension before FACS analysis using antibodies against human CD133 and CD15.
Figure 2
Figure 2
Elevated expression of tumor-associated antigens in human glioblastoma-derived stem-like cells. Representative tumor-associated antigen expression in glioblastoma multiforme cancer stem-like cells (GBM1-5). Cancer stem-like cells single cell suspension was stained with specific monoclonal antibodies to each tumor-associated antigens and an isotype-matched control antibody, followed by FACS analysis. Results are given as the ratio of antigen expression levels in CSCs to those in daughter cells.
Figure 3
Figure 3
Glioblastoma-derived stem-like cells express class I MHC molecules on the cell surface. A subset of CSCs expresses both class I and class II MHC molecules. Single cell suspension of neural stem cells (NSC) and cancer stem-like cells (CSC3-5) was stained with specific antibody to class I MHC (HLA-A,B,C), class II MHC (HLA-DR) and an isotype-matched control antibody, followed by FACS analysis. Results were representative of three independent experiments.
Figure 4
Figure 4
Dendritic cell vaccination using cancer stem-like cell antigens generates antigen-specific Th1 response. A&B. IFN-γ release by antigen-specific CD8+ cytotoxic T lymphocytes recognizing tumor-associated antigens HER-2, TRP, CD133, IL13Rα2, and Sox2 (A) and IFN-γ release by CD8+ CTLs after vaccination with different CSCs (B). 1 × 105 clonal CTL cells were incubated with 1 × 105 HLA-matched tumor cells as compared with CTL cells incubated with 1 × 105 control cells. ** p< 0.01 (compared with control groups). C. Assessment of tumor-associated antigen-specific T cell clones by HLA/peptide tetramer staining. HER-2, TRP, CD133, IL13Rα2, and Sox2-specific CTL clones (1 × 105 cells) were stained with phycoerythrin-conjugated peptide/HLA tetramer at room temperature for 30 min. Cells were then incubated with antibody against CD8 for 30 min at 4°C. Cells were then examined by fluorescence-activated cell-sorting analysis using 10,000 events/sample. Generation of antigen-specific CTL is positively correlated with Th1 response as indicated by IFN-γ production (coefficient R2=0.86).
Figure 5
Figure 5
Therapeutic effect of neurosphere-pulsed DC against intracranial 9L tumor in adult rats. Rats were injected intracranially with 25000 9L glioma cells on day 0 and were vaccinated s.c. on days 7, 14, and 21 with different tumor antigen pulsed dendritic cell vaccines: control (DC only), monolayer, daughter cells, and neurospheres (n=10 for each group). Kaplan-Meier survival curve showed that rats treated with 9L neurosphere lysate-pulsed DC have longer survival than the other groups (** p=0.0015). The surviving animals with CSC vaccination were sacrificed after 70 days and slowly growing brain tumors were detected.
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