The increase in MHCI expression on tumor cells is dependent on cell-cell contact between NSCs and tumor cells. The increase in MHCI expression on tumor cells is dependent on cell-cell contact between NSCs and tumor cells. (A) MC57, MCA102, B16 and TEC tumor cells were cultured in the absence (control) or presence of IFN-γ for two days. Then, mRNA was isolated, cDNA synthesized using primers specific for Db, Kb, tapasin (tapa) or actin, and the PCR products were separated in agarose gels. DNA fragments were revealed by BET. MCA102 and B16 cells were MCA102gp and B16gp cells, respectively. (B) B16gp cells (2.5 x 104) were cultured for 3 days alone or with 300 pg/ml IFN-γ in 3 ml wells as negative (column 1) or positive controls (column 2). Alternatively, B16gp cells were cultured with NSCs (10 x 106) in 3 ml wells (column 3), or B16gp cells were cultured in the wells and NSCs in the insert (column 4) and vice versa (column 5). B16gp cells were also cultured in the well with a mixture of B16gp and NSCs in the insert (column 6). The data in columns 1-6 are referred to as groups 1-6 in the text. H-2Db expression of the tumor cells in the wells was measured by flow cytometry using FITC-labeled anti-Db mAb (28.14.4S). (C) B16gp (squares), MCA102gp (circles) and TEC-427.1 cells (triangles) were cultured alone (open symbols) or with IFN-γ (100 pg/ml) (closed symbols) for 3 days. The tumor cells were then harvested, washed and assayed for sensibility to the cytotoxic activity of alloreactive T cells (BALB/c anti-B6 T cell line) at different target:effector cell ratios. The data are means of quadruplicates, and variations did not exceed 10% (not shown). (D) NSCs from wild-type (a) or IFN-γ-/- deficient mice (b) were co-cultured for 3 days with B16gp cells at ratios of 600:1, 400:1, 200:1 or 100:1. The B16gp cells were then harvested and H-2Db expression was measured by flow cytometry. Tumor cells were either cultured in medium alone (negative control) or stimulated with 100 pg/ml of IFN-γ (positive control) (c). (E) NSCs (3 x 106/ml) were cultured in quadruplicates with MHCIlow TEC-427.1 (5 x 104/ml) tumor cells for four days. After 3, 6, 9, 12, 18, 24, 36, 48, 60, 72, 84, and 96 hours 1 ml of supernatant was isolated. These supernatants were analyzed for IFN-γ content by ELISA (open diamonds) and for their capacity to induce an increase in MHCI expression by flow cytometry on TEC-427.1 cells with FITC anti-Db mAb (MFI on quadruplicates were less than 6%) on MHCIlow tumor cells (filled diamonds). Standard deviations of quadruplicates of IFN-γ determinations did not exceed 10%. Four experiments were performed with identical results. Bent Rubin et al. Cancer Immun 2008;8:14 Copyright © 2008 by Bent Rubin