Abstract
Abstract BACKGROUND Glioblastomas (GBMs) are aggressive brain tumors with strong immunosuppressive properties. In epithelial cancers, mesenchymal cells expressing fibroblast activation protein (FAP) play an important role in modulating the T cell response. We have recently shown that FAP+ mesenchymal cells are present in human GBMs. The aim of this study was to determine their effect on T cell abundance and function. MATERIAL AND METHODS GBM tumors (16 samples) were mechanically and enzymatically dissociated and analyzed for immune cell subpopulations by flow cytometry. Expression of T cell related genes was analyzed by qRT-PCR in GBMs selected based on FAP protein concentration determined by ELISA (14 FAP high [upper tercile], 16 FAP low [lower tercile]). Immunohistochemistry (IHC) was used to compare the number of CD3+, CD4+, and CD8+ T cells in GBMs with high (upper tercile, n=21) and low abundance of FAP+ stroma (lower tercile, n=20). Co-localization of FAP+ mesenchymal cells and CD3, PD-L1 and PD-L2 was evaluated by immunofluorescence in frozen sections. 8 FAP+ mesenchymal and 3 glioblastoma stem-like cell cultures were derived from human GBMs and PD-L1 and PD-L2 expression was determined using ELISA. Peripheral blood mononuclear cells were isolated from healthy donors´ buffy coats. Proliferation of stimulated T cells in conditioned media from FAP+ mesenchymal cells was analyzed by flow cytometry. RESULTS The percentage of T cells in CD45+ cells from GBMs positively correlated with FAP expression. Expression of the T cell inhibitory molecule PD-L2 was higher in FAP high GBMs. Immunohistochemisty revealed that the numbers of CD3+ and CD8+ T cells were higher in GMBs with a high abundance of FAP+ stroma and T cells were frequently in close proximity to FAP+ mesenchymal cells, which were often PD-L2 positive. In vitro, FAP+ mesenchymal cells expressed more PD-L2 than glioblastoma stem-like cells. T cell proliferation was decreased after exposure to conditioned media from FAP+ mesenchymal cells. CONCLUSION FAP+ mesenchymal cells influence T cell abundance in GBMs and may affect T cell functions by limiting their proliferation via soluble factors or cell-cell contact. Acknowledgment: This work was supported by the National Institute for Cancer Research (LX22NPO5102), the Center for Tumor Ecology (CZ.02.1.01/0.0/0.0/16_019/0000785), Charles University project GAUK 365022 and Cooperatio Program Oncology and Haematology“.
Published Version
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