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. 2009 Dec 1;69(23):8886-93.
doi: 10.1158/0008-5472.CAN-09-2687. Epub 2009 Nov 10.

Recognition and killing of brain tumor stem-like initiating cells by CD8+ cytolytic T cells

Christine E Brown  1 , Renate Starr, Catalina Martinez, Brenda Aguilar, Massimo D'Apuzzo, Ivan Todorov, Chu-Chih Shih, Behnam Badie, Michael Hudecek, Stanley R Riddell, Michael C Jensen
Affiliations

Recognition and killing of brain tumor stem-like initiating cells by CD8+ cytolytic T cells

Christine E Brown et al. Cancer Res. .

Abstract

Solid tumors contain a subset of stem-like cells that are resistant to the cytotoxic effects of chemotherapy/radiotherapy, but their susceptibility to cytolytic T lymphocyte (CTL) effector mechanisms has not been well characterized. Using a panel of early-passage human brain tumor stem/initiating cell (BTSC) lines derived from high-grade gliomas, we show that BTSCs are subject to immunologic recognition and elimination by CD8(+) CTLs. Compared with serum-differentiated CD133(low) tumor cells and established glioma cell lines, BTSCs are equivalent with respect to expression levels of HLA class I and ICAM-1, similar in their ability to trigger degranulation and cytokine synthesis by antigen-specific CTLs, and equally susceptible to perforin-dependent CTL-mediated cytolysis. BTSCs are also competent in the processing and presentation of antigens as evidenced by the killing of these cells by CTL when antigen is endogenously expressed. Moreover, we show that CTLs can eliminate all BTSCs with tumor-initiating activity in an antigen-specific manner in vivo. Current models predict that curative therapies for many cancers will require the elimination of the stem/initiating population, and these studies lay the foundation for developing immunotherapeutic approaches to eradicate this tumor population.

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Figures

Figure 1
Figure 1. In vitro characterization of expanded tumor spheres and serum-differentiated cells expanded from primary high grade glioma specimens
(A) TS and DIF cells were analyzed by flow cytometry using anti-CD133 (grey) or isotype control antibody (solid line), and percent CD133+ cells are indicated. (B) Western blots detecting CD133, Olig2, GFAP and ß-III tubulin in TS and DIF cells. Fold change in expression under serum growth conditions was normalized to actin and is indicated. (C) Quantitation (ng/ml) of VEGF secreted by TS and DIF cells. (D) Comparison of PBT003 scp4 TS orthotopic tumor histology (top panels) with primary human patient tumor histology (bottom panels) showing similar phenotypic appearance, with intrinsic diagnostic features of glioblastoma, including pseudopalisading necrosis (left panel outlines), prominent microvasculature (circle and oval); and similar interactions with non-neoplastic host tissue elements, such as perineuronal satellitosis (PS), perivascular growth (PV), and marked leptomenigeal invasion (arrows).
Figure 2
Figure 2. Flow cytometric analysis of MHC I, MHC II, and ICAM-1 expression
(A) TS (top panels) and DIF cells (bottom panels) and (B) freshly dispersed glioma tumors (FDT) were analyzed by flow cytometry using the indicated antibodies (black histograms) or isotype controls (solid line). Percent positive cells are indicated. (C) FDT cells were double stained for CD133 versus HLA-ABC, HLA-DR, CD54 (ICAM-1), or CD31 expression. N.D. not done.
Figure 3
Figure 3. CMV-specific CTL kill pp65-peptide loaded PBT008 tumor stem cells equivalent to differentiated and established glioma cell lines
(A) LCA and (B) CRA measuring the lysis of CMV pp65 peptide (NLVPMVATV) versus control influenza M1 peptide (GILGFVFTL) loaded PBT008-GL+ (EGFP-ffLuc+) TS, serum-differentiated PBT008-GL+ (DIF), or U251T-GL+ cells by CD8+ CMV pp65-specific CTL at increasing E:T ratios (mean ± S.D. of n=6 wells). *, p < 0.006 when comparing M1 versus pp65 peptide loaded targets using unpaired Student's t-test. (C) IFN-γ and TNF-α produced by CTL after co-culture with specified peptide loaded tumor targets. (D) Flow cytometry-based killing assay monitoring the loss of viable CD133high, CD133low, or total PBT008 TS cells at increasing E:T ratios. Inset shows PBT008 TS CD133 expression, and the percent CD133high (48%) and CD133low (51%).
Figure 4
Figure 4. Perforin-dependent killing by CMV-specific CTL equivalently targets pp65-peptide loaded PBT003 TS and DIF cells
(A) Lysis of CMV pp65 peptide (QYDPVAALF) loaded PBT003-GL+ (EGFP-ffLuc+) TS or DIF cells by the CD8+ pp65-specific CTL at increasing E:T ratios was determined by LCA (mean ± S.D. of n=6 wells). *, p < 0.0004 when comparing +/− pp65 peptide using unpaired Student's t-test. (B) IFN-γ and TNF-α produced by CTL after co-culture with specified tumor targets. (C) Flow cytometric detection of CD107a on CD8+ CTL after co-culture with tumor cells as in (A) at a 2:1 E:T ratio in the presence (lower panels) and absence (upper panels) of CMV peptide. (D) Inhibition of LCA-measured killing of pp65-loaded PBT003-GL+ scp4 TS and DIF tumor targets at a 10:1 E:T ratio upon pre-incubation of CD8+ pp65-specific CTL with increasing concentrations of CMA. At all CMA concentrations p < 0.003 when compared to no CMA using unpaired Student's t-test.
Figure 5
Figure 5. Tumor stem cells and serum-differentiated glioma cells expressing endogenous pp65 antigen are equivalently recognized and lysed by CD8+ CTL
(A) Schematic of EGP:ffLuc-2A-pp65 bicistronic construct endowing forced co-expression of the EGFP:ffLuc reporter and pp65 antigen. 3×G, 3 glycine linker; 2A, self-cleaving peptide. (B) Lysis of tumor cells expressing either EGFP:ffLuc alone (GL) or EGFP:ffLuc and the CMV-pp65 antigen (GL-pp65) by HLA-matched CD8+ CMV-specific CTL was determined by LCA (mean ± S.D. of n=6 wells). *, p < 0.045 when comparing GL to GL-pp65 expressing targets using unpaired Student's t-test.
Figure 6
Figure 6. CMV-specific CTL ablate the tumor initiation potential of pp65 expressing TS
2×105 PBT003 (scp4) TS expressing either EGFP:ffLuc (TS-GL) or EGFP:ffLuc and the CMV-pp65 antigen (TS-GL-pp65) were co-injected i.c. with 2×106 pp65-specific CTL or PBS into NOD/scid mice; n = 6 mice per group. (A) Flow cytometric analysis of TS-GL, TS-GL-pp65 with only 57.2% of the cells expressing the transgenes (+/−) or TS-GL-pp65 with 91.9% of the cells expressing the transgenes (++). (B) Mean biophotonic flux of the i.c. tumors ± S.E. over time was determined by Xenogen imaging. *, p = 0.017 when comparing TS-GL-pp65+/+ tumors that had been co-injected with CTL vs. PBS at day 47 using an unpaired Student's t-test. **, p = 0.036 when comparing TS-GL-pp65+/− tumors that had been co-injected with CTL vs. PBS at day 48 using an unpaired Student's t-test. (C) Representative biophotonic images of mice from each group at day 47. (D) Tiled images of horizontal brain sections from representative mice that had received TS-GL-pp65 cells with PBS (top panels) or with CTL (bottom panels) and were stained with H&E (left), or for the B23 human cell marker (middle two) or the CMV pp65 antigen (right) by IHC. Anti-B23 and anti-pp65 IHC near the injection site is shown to the right.
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