Figure 2.

IFN-γ-mediated activities in vitro. A) Eleven NB cell lines were tested for expression of MHC class I by flow cytometry. Solid black lines, anti-MHC class I on untreated cells; gray lines, anti-MHC class I on cells treated with IFN-γ (100 U/ml for 48 hrs); filled histograms, isotype control antibody. B) Diagnostic PBMC from 2 HLA-A2⁺ patients were stimulated with CD40-B cells loaded with survivin mRNA or GFP mRNA, and analyzed after 14 days in culture. Tumor targets were MHC class I negative NLF cells (circles) or MHC class I negative SY5Y (diamonds). Tumor targets were either untreated (open symbols) or treated with IFN-γ (filled symbols). Both cell lines upregulated MHC class I following IFN-γ but only NLF cells were HLA-A2⁺. Both cell lines were survivin positive by RT-PCR. C) Three MHC Class I negative NB cell lines were tested for upregulation of expression of MHC class I by IFN-γ in vivo. NOG mice bearing established NB cell line tumors were injected subcutaneously adjacent to the tumor with recombinant human IFN-γ or PBS daily for 3 injections (30,000 IU/injection) and tumors harvested on day 4. Single cell suspensions were prepared and examined by flow cytometry for expression of HLA-A,-B,-C. Solid black lines, anti-MHC class I on untreated cells; dashed lines, anti-MHC class I on cells treated with IFN-γ; filled histogram, isotype control antibody on untreated cells; solid gray lines, isotype control antibody with IFN-γ.