126 Tumor biology & interaction with the immune system
078 Maccalli | Tumor biology & interaction with the immune system Definition of the immunological properties of cancer stem cells isolated from human glioblastoma Cristina Maccalli 1 , Tiziano Di Tomaso 1 , Ena Wang 2 , Stefania Mazzoleni 3 , Gloria Sovena 1 , Soldano Ferrone 4 , Rossella Galli 3 , Francesco M. Marincola 2 , Giorgio Parmiani 1 1 Unit of Immuno-Biotherapy of Solid Tumors, San Raffaele Foundation Scientific Institute, Milan, Italy 2 Department of Tranfusion Medicine, National Institutes of Health, Bethesda, MD, USA 3 Neural Stem Cell Unit, San Raffaele Foundation Scientific Institute, Milan, Italy 4 Hillman Cancer Center, University of Pittsburgh Cancer Institute, Pittsburgh, USA The main objectives of our study is to assess whether cancer stem cells (CSCs) isolated from glioblastoma multiforme (GBM) patients can be exploited as source of antigens to elicit T cell-mediated immune responses and their validation to design immunotherapeutic protocols for GBM. We have characterized, by immunofluorescence and cytofluorimetric or confocal microscopy analysis, the immune profile of 14 CSC and of 7 FBS tumor line pairs isolated from GBM samples. Moreover, PBMCs isolated from GBM patients were in vitro stimulated with autologous CSCs and the specific reactivity of T lymphocytes against GBM CSCs was evaluated by IFN-g, Granzyme B release (ELISPOT) or CD107a mobilization. We have assessed that both GBM CSC and their FBS-cultured non-CSC pairs showed a low immunogenic profile (MHC, NKG2DL and APM molecules). Up-regulation of most of these molecules was induced by IFNs or 5-Aza CdR, though more efficiently in FBS than in CSCs. Notably, 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 pre-treated with IFN-β. However, TH2-mediated responses with more efficient recognition of FBS tumor cell than CSCs, could be found in 3 additional GBM patients. One key finding of this study is that CSCs but not FBS cells inhibit allogeneic T cell proliferation. This immune-inhibitory activity mediated by GBM CSCs was not dependent on the secretion of TGFβ1 or 2, that were preferentially down-modulated in CSC lines, nor on IL-10 and IL1-3 that were undetectable in the supernatant of these cell lines. Expression of the indoleamine 2,3dioxygenase (IDO), a molecule implicated in the generation of immune tolerance, was detected in both CSCs and FBS tumor cell lines with high increase following IFN-g treatment of the cells. Furthermore, we found that both CSCs and FBS tumor cells expressed immune response inhibitory molecules, such as CTLA-4, PD-1 and PDL-1. Furthermore, a differential gene signature that was confirmed at the protein levels for some immunological-related molecules was also found between CSC and FBS lines. Moreover, this latter analysis provided information of some candidate molecules associated with CSCs that we are functionally investigating. Altogether, these results indicate a lower immunogenicity and higher suppressive activity of GBM CSC compared to autologous FBS lines and the need to identify immunomodulatory agents that can efficiently restore the expression of immunogenic molecules on CSCs. 127