Previous studies indicated that B7-H4,the youngest B7 family,negatively regulates T cell-mediated immunity and is significantly overexpressed in many human tumors.Tumor stem cells are purported to play a role in tumor renewal and resistance to radiation and chemotherapy.However,the link between B7-H4and tumor stem cells is unclear.In this study,we investigated B7-H4 expression in the medium of human glioma U251 cell cultures.Immunofluorescence results showed that U251 cells cultured in serum-free medium(supplemented with 2%B27,20 ng/mL epidermal growth factor,20 ng/mL basic fibroblast growth factor)maintained stem-like cell characteristics,including expression of stem cell marker CD133 and the neural progenitor cell markers nestin and SOX2.In contrast,U251 cells cultured in serum-containing medium highly expressed differentiation marker glial fibrillary acidic protein.Flow cytometry analysis showed serum-free medium-cultured U251 cells expressed higher intracellular B7-H4 than serumcontaining medium-cultured U251 cells(24%-35%vs.8%-11%,P<0.001).Immunofluorescence in purified monocytes from normal human peripheral blood mononuclear cells revealed moderate expression of B7-H4 after stimulation with conditioned medium from U251 cells cultured in serum-containing medium.Moreover,conditioned medium from U251 stem-like cells had a significant stimulation effect on B7-H4expression compared with serum-containing conditioned medium(P<0.01).Negative costimulatory molecule B7-H4 was preferentially expressed in U251 stem-like cells,and conditioned medium from these cells more effectively induced monocytes to express B7-H4 than conditioned medium from U251 cells cultured in the presence of serum.Our results show that U251 stem-like cells may play a more crucial role in tumor immunoloregulation with high expression of B7-H4. Previous studies indicated that B7-H4, the youngest B7 family, negatively regulates T cell-mediated immunity and is significantly overexpressed in many human tumors. Tumor stem cells are purported to play a role in tumor renewal and resistance to radiation and chemotherapy. However, the link between B7-H4 and tumor stem cells is unclear. In this study, we investigated B7-H4 expression in the medium of human glioma U251 cell cultures. Immunofluorescence results showed that U251 cells in serum- free medium (supplemented with 2% B27 , 20 ng / mL epidermal growth factor, 20 ng / mL basic fibroblast growth factor) maintained stem-like cell characteristics, including expression of stem cell marker CD133 and the neural progenitor cell markers nestin and SOX2.In contrast, U251 cells cultured in serum -containing medium highly expressed differentiation marker glial fibrillary acidic protein. Flow cytometry analysis showed serum-free medium-cultured U251 cells showed higher intracellular B7-H4 than serumcon (24% -35% vs 8% -11%, P <0.001). Immunofluorescence in purified monocytes from normal human peripheral blood mononuclear cells revealed moderate expression of B7-H4 after stimulation with conditioned medium from U251 conditioned medium from U251 stem-like cells had a significant stimulation effect on B7-H4expression compared with serum-containing conditioned medium (P <0.01) .Negative costimulatory molecule B7-H4 was preferentially expressed in U251 stem-like cells, and conditioned medium from these cells more effectively induced monocytes to express B7-H4 than conditioned medium from U251 cells cultured in the presence of serum. Our results show that U251 stem-like cells may play a more crucial role in tumor immunoloregulation with high expression of B7-H4.