Immunobiological characterization of cancer stem cells isolated from glioblastoma patients

Abstract

Purpose: Cancer stem cells (CSC) have been isolated from human tumors, including glioblastoma multiforme (GBM). The aims of this study were the immunobiological characterization of GBM CSCs and the assessment of whether these cells represent suitable targets for immunotherapy.

Experimental design: GBM CSC lines and their fetal bovine serum (FBS)-cultured non-CSC pair lines were generated and examined by flow cytometry for expression of known tumor antigens, MHC-I and MHC-II molecules, antigen-processing machinery components, and NKG2D ligands. In addition, immunogenicity and immunosuppression of such cell lines for autologous or allogeneic T lymphocytes were tested by cytokine secretion (ELISPOT) or proliferation (carboxyfluorescein diacetate succinimidyl ester) assays, respectively.

Results: Both GBM CSC and FBS lines were weakly positive and negative for MHC-I, MHC-II, and NKG2D ligand molecules, respectively. Antigen-processing machinery molecules were also defective in both cell types. Upregulation of most molecules was induced by IFNs or 5-Aza deoxycytidine, although more efficiently in FBS than in CSCs. Patient T-cell responses, mediated by both TH1 and the TH2 subsets, against autologous CSC could be induced in vitro. In addition, CSC but not their paired FBS tumor lines inhibited T-cell proliferation of healthy donors. Notably, a differential gene signature that was confirmed at the protein levels for some immunologic-related molecules was also found between CSC and FBS lines.

Conclusions: These results indicate lower immunogenicity and higher suppressive activity of GBM CSC compared with FBS lines. The immunogenicity, however, could be rescued by immune modulation leading to anti-GBM T cell-mediated immune response.

Fig. 1

Reactivity of 070104 T lymphocytes against autologous GBM CSCs. PBMCs isolated from GBM patient 070104 were stimulated in vitro with autologous irradiated CSCs and, following 3 wk of culture, the reactivity against autologous or allogeneic GBM CSCs was assessed by IFN-γ release (ELISPOT assay). Cytokine secretion was determined following stimulation of T cells with autologous CSCs (black columns), autologous CSCs pretreated with anti-MHC class I (W6/32) or anti-MHC class II (L243) mAbs (gray and dotted white columns, respectively), autologous CSC in vitro cultured for a few weeks in the presence of FBS (gray dotted bar), autologous FBS-cultured CSCs pretreated with anti-MHC class I (W6/32) or anti-MHC class II (L243) mAbs (small grid and gray bars, respectively), allogeneic HLA-mismatched CSCs (0627 and 070112; black squared bars, respectively) and, as positive control with OKT3 mAb (dark gray bar). As shown in the figure, three independent T-cell lines with specific recognition of autologous CSCs were in vitro isolated (#2, 3, and 10). Data are expressed as number of spot per 4 × 10⁴ cells. Results represent averages of triplicates with an SD of ≤10%; statistical analysis of differences between means of IFN-γ released by T cells was done by two-tailed t test.