In this study, we exploited the availability of a panel of CSCs and, in some cases, of autologous FBS tumor cells isolated from GBM patients to characterize their immune profile and to explore the possibility of targeting CSCs by raising specific T cell–mediated immune responses. Our results show that GBM-derived CSCs expressed MHC class I molecules, although at lower level than FBS tumor cells (Supplementary Fig. S2
; ). These molecules were significantly upregulated by IFN-α or IFN-γ treatment; the increase of MHC class I being higher in FBS tumor cells than in their CSC pairs. Conversely, lack or the negligible expression of MHC class II molecules was found for both CSCs and FBS tumor cells that could not be restored, except for one CSC line (070104), by IFNs (Supplementary Fig. S2
; ). In addition, CSCs and FBS tumor cells lacked or expressed low level of NKG2DLs, and only in few cases, these ligands could be upmodulated by treatment with IFN-α (Supplementary Fig. S2
). Furthermore, defective expression of an array of APM molecules, except for γ, Calreticulin, and LMP2/7, was commonly found in both CSC and FBS tumor cells, and the IFN treatment could modulate the expression of some but not other molecules (e.g., LMP10, TAP1, MB1, ERp75, and Tapasin). These results are in line with the previously reported defective expression of MHC and APM molecules in a variety of human tumors (25
), indicating that low efficiency in antigen processing and presentation occur also in GBM and, interestingly, in CSC isolated from this tumor type.
Hitherto, these results confirm the low immunogenicity of both GBM-derived CSCs and FBS tumor cells, which may render them resistant to the T cell–mediated immune reactions. Nevertheless, incubation of these cells with IFNs or the demethylating agent 5-Aza-2′-deoxycytidine can partially restore the expression of MHC, APM, and NKG2DLs, although to a higher level in FBS tumor cells than in CSCs, thus suggesting, as shown in a large number of melanoma cell lines (43
), that different mechanisms can affect alterations in antigen processing and presentation by GBM CSCs and FBS tumor cells. These results are in line with previous observations showing that GBM CSC, isolated on the basis of the expression of CD133 molecule, as well as CD133−
cells, failed to express detectable level of MHC class I and natural killer (NK) cell activatory ligands, leading to resistance by these cells to NK-mediated lysis and showing that IFN-γ treatment of these cells could increase their susceptibility to NK cell activity (44
). Notably, we found no or weak in vivo
expression of MHC, APM, and NKG2DLs by GBM tissues, thus confirming the results obtained for GBM cell lines and indicating that these cell lines are indeed representative of the original neoplastic lesions.
Furthermore, among the 469 genes that resulted, by the gene profile analysis, differentially expressed in GBM CSCs versus FBS tumor cells, the downmodulation of some immunologic-related genes, such as proteasome components (proteasome maturation protein and the proteasome activator subunit 1) IFN-γ and IFN-γR1, correlate with our observations that the defective expression of MHC and APM molecules can be more pronounced in GBM CSCs. Moreover, reduced 26S proteasome activity has been found as a general feature of CSCs isolated from GBM and breast cancer (45
Interestingly, IL-6 and IL-8 were also found downmodulated in CSCs versus FBS tumor lines, correlating with the results obtained at protein level (Supplementary Fig. S6
One key finding from our study is that T-cell responses, both CD4+
– and CD8+
–mediated, against GBM could be raised in one GBM patient by stimulating in vitro
PBMCs with autologous CSCs pretreated with IFN-γ (). In addition, CD8+
T cell–mediated cytotoxic activity, evaluated by CD107 mobilization and Granzyme B secretion, against autologous CSCs could be elicited, although the TAA recognized by these T cells still need to be defined. Of note, this anti-CSC T cell–mediated activity correlated with the expression of MHC class II molecules by IFN-treated CSCs (). Notably, it was recently shown that CMV-specific CTLs could exert cytotoxic activity, showing consistent results with both CD107a and 51
Cr-release assays, against CSC-like cells isolated from GBM (46
). However, most TH2-mediated (detected by IL-5 release) responses against both CSCs and FBS tumor cells could be found in additional three GBM patients, and these CD4+
T cells were more efficient in recognizing FBS tumor cell than CSC (). Of note, an expansion and recruitment of CD4+
T cells subset with TH2 cytokine profile in tumor-infiltrating lymphocytes of GBM patients has been reported as a result of local tumor suppression of cell-mediated immune responses (47
). Furthermore, we found that both CSCs and FBS tumor cells expressed immune response inhibitory molecules, such as CTLA-4, PD-1, and PDL-1, which could inhibit T-cell activation and proliferation following their encountering with GBM cells. The expression of B7 family members have been described in brain tumor cells, including their CSCs, with implications for the T cell–mediated immunesurveillance (48
). In addition, CTLA-4 blockade reversed glioma-induced changes to the T-cell compartment and enhanced antitumor immunity in an experimental brain tumor model (50
Interestingly, we could show that CSC but not FBS cells inhibit allogeneic T-cell proliferation (). This suggests that CSCs can exert immunomodulating functions similarly to neural stem cells as shown by Kim et al. (51
). Our results showed that this immune-inhibitory activity mediated by GBM CSCs was not dependent on the secretion of some immunosuppressive factors such as TGFβ-1 or TGFβ-2, which were preferentially downmodulated in CSC lines (Supplementary Fig. S6
), or IL-10 and IL1-3, which was lacking in the supernatant of these cell lines (data not shown). Therefore, CSCs could secrete factors or express negative regulatory molecules that need to be identified and that could result in immunosuppressive effects on T-cell responses against GBM.
Our study represents the first detailed analysis of the immunologic features of GBM-derived CSCs and their autologous FBS tumor cells. Although we found a lack or weak expression of molecules involved in APM and antigen presentation on CSCs, immunogenicity of these cells could be rescued by either IFNs or demethylating agent treatment more efficiently in FBS than CSC cells. Interestingly, both GBM-derived CSCs and FBS tumor cells can elicit autologous antigen-specific T cell–mediated immune responses.
Our findings are supported by the results of two different groups showing that the CSCs isolated from GBM can represent a useful source of TAAs in dendritic cell (DC) vaccination–based immunotherapy, including human and mouse models (52
), suggesting that the usage of efficient APCs is needed to elicit anti-GBM CSC T cell–mediated immune responses.
Altogether, immunobiological differences between GBM CSC and autologous FBS tumor lines emerge for several phenotypic and functional aspects. A general explanation for such differences may lie in the less differentiated stage of CSC compared with the more differentiated FBS tumor cells as can be seen from differences in the expression of neural stemness–related markers (e.g., nestin, SOX2, and S100A4/A6). Such a lower differentiation stage of CSC appears to be associated with a weaker expression of molecules (MHC-I, NKG2D) that are crucial for T-cell and NK cell recognition of antigens and to a more accentuated immunosuppressive activity.
Both GBM CSC and FBS tumor cell lines homogeneously displayed, by confocal microscopy, high levels of the colorectal cancer–associated antigen COA-1 (24
) and of survivin (Supplementary Fig. S7A and C, and B and D
, respectively). Surprisingly, all these cells failed to express some of the known TAAs, such as gp100, MAGE, NY-ESO-1, and IL-13 receptor α2 previously described in GBM (data not shown; refs. 3, 6, 7), possibly resulting from immune escape and/or in vitro
culture selection of these GBM cell subpopulations. Therefore, suggesting that survivin, COA-1 and SOX2 (see Supplementary Fig. S7
and ) are candidate GBM CSC–associated target molecules recognizable by patient T cells because each of these antigens has been shown to contain epitopes recognized by T lymphocytes (5
Notably, the high throughput gene profile analysis showed that differentially expressed genes in CSC versus FBS tumor cells occurred and the further functional analysis of these results will contribute in dissecting the involvement of some of these gene products in CSC proliferation and/or therapeutic resistance as well as in determining whether they can represent candidate TAAs and targets for immunesurveillance. Of note, a recently published study showed that genomic and mRNA profiling of GBM CSCs allowed the identification of CSC-associated candidate molecules that can play a role in the pathogenesis of this tumor and in the clinical outcome of GBM patients (19
Hitherto, our results have implications for immunotherapy of GBM patients and indicate the need to identify agents that can efficiently rescue the immunogenicity of CSCs to be targeted with immunobase therapeutc interventions.