Figure 1.
Expression of MHC-I, IRF1 and IRF2 in NB cell lines.
A, flow cytometry analysis of surface MHC-I expression in NB cell lines using W6/32 mAb (grey lines). Shaded histograms, negative controls stained with isotype-matched primary antibody. B, immunoblot analysis of IRF1 and IRF2 in NB cell lines. Equal amounts of whole-cell extracts and nuclear extracts, as indicated, were resolved by SDS-PAGE, immunoblotted and probed with specific antibodies. ERp57 and PCNA were used for normalization. Positive and negative IRF1 and IRF2 controls, as well as densitometric and statistical analysis of WB bands are shown in Fig. S1. C, qRT-PCR analysis of mRNAs from different NB cell lines. 18S RNA was used for normalization. Significant differences between the 3 MHC-I-expressing NB cells and the 5 MHC-I-low NB cells of mRNA expression separately averaged were evaluated as described in Materials and Methods. IRF1: P<0,001; IRF2: P = 0,09; ± SD of triplicate assays. Data shown in the panels A to C are representative of at least 3 independent experiments.
Figure 2.
Expression of MHC-I, IRF1, IRF2 and the NF-kB p65 subunit in primary NB lesions.
Immunohistochemistry of human NB tissue sections with Abs to MHC-I (A, E), IRF1 (B, F), IRF2 (C, G) or NF-kB p65 subunit (D, H). Visualized with diaminobenzidine (DAB; brown), nuclei counter-stained with haematoxilin (blue). IRF1, IRF2 and NF-kB p65 are strongly expressed in the nuclei of mature ganglion cells (arrows), endothelial cells, lymphocytes and stroma cells in the well-differentiated MHC-I-positive ganglioneuroblastoma (A-D), and weakly expressed in the MHC-I-negative neuroblastic cells (arrowhead), i.e. undifferentiated stroma-poor NB (E-H). In E-H, positive staining of benign cells, including lymphocytes and macrophages. NF-kB p65-positive staining of the fibrillary network in H is evident. Original magnification, x40. Scale bars 30 µm. Data shown are representative of 10 stroma-rich and 10 stroma-poor NB tissue sections.
Figure 3.
IFN-γ restores IRF1, MHC-I and APM components in NB cell lines.
A, whole-cell lysates of NB cell lines cultured for 48 hours in the presence and absence of IFN-γ were resolved by SDS-PAGE and Western blotted with specific antibodies. ERp57 was used as loading control. Densitometric and statistical analysis of WB bands are shown in Fig. S4. B, flow cytometry analysis with mAb W6/32 of surface MHC-I expression of NB cells grown, as above, for 48 hours in the presence and absence of IFN-γ. Untreated cells (grey lines), treated cells (black lines) and isotype-matched negative controls (shaded histograms) are shown. Data shown in panels A and B are representative of 3 and 5 independent experiments, respectively.
Figure 4.
IRF1 and p65 synergistically enhance MHC-I and APM components in NB cell lines.
A, immunoblotting of cell extracts from three NB cell lines left untransfected (none) or single- and double-transfected, as indicated, with the control empty vector (pcDNA3) or vectors expressing IRF1, IRF2, and NF-kB p65. ERp57 was used as loading control. Densitometric and statistical analysis of WB bands related to the cells transfected with pcDNA3, or p65, or IRF1 and p65 are shown in Fig. S5. B, flow cytometry analysis of surface MHC-I expression in the same NB cell lines (indicated by different colors) with mAb W6/32. Isotype-matched negative controls are displayed as shaded histograms. Data shown in panels A and B are representative of 5 and 8 independent experiments, respectively.
Figure 5.
Double IRF1/p65 transfection renders NB cells susceptible to killing by specific cytotoxic T cells.
A, RT-PCR analysis of tumor antigens in NB cell lines. The melanoma cell line HO-1 was included as positive control. GAPDH gene expression was used for normalization. B, the SH-SY5Y cell line (5Y) grown in the presence of IFN-γ (5Y-IFN-γ) for 48 hours or co-transfected with IRF1 (5Y-IRF1) and the NF-kB p65 subunit (5Y-IRF1-p65, and 5Y-p65 respectively) or empty vector (5Y-pcDNA3) was assayed by either MHC-I expression (Fig. S6B) by flow cytometry or as targets to HLA-A1-restricted/MAGE-A3-specific CTLs at the indicated effector:target (E:T) ratios in a standard 51Cr-release assay. Statistically significant differences are indicated (*, P<0.002, **, P<0.0000002). Mean ± SD of three experiments is shown. Data shown in A and B are representative of 3 experiments.