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Fig. 1. Peptide-MHCII complexes are formed and presented to T cells from a cytosolically expressed as well as from a cytosolically delivered protein. (A) BMC-2 cells in 6-well dishes were transfected with 10 µg of either E_52-68-eGFP or eGFP (control) DNA and stained 24 hours later with the Y-Ae antibody. Fluorescence of GFP and Y-Ae was analysed by flow cytometry. Data are shown as twocolour plots and as single-parameter graphs for Y-Ae fluorescence of appropriately gated eGFP+ and eGFP- cells as indicated. Shaded area under curve, untransfected cells; thin lined curves, eGFP transfected cells; thick lined curves, E_52-68-eGFP transfected cells. (B) BMC-2 cells pulsed cytosolically (via osmotic pinosome lysis method) or exogenously via the fluid phase with 5 mg/ml GST E_52-68-myc fusion protein were stained with Y-Ae after 1 hour and analysed by flow cytometry. Shaded curve, unstained cells; thin line, unloaded cells; thick line, exogenously loaded cells; grey line, cytosolically loaded cells. (C) Responses of the 1H3.1 T cell line to titrating numbers of BMC-2 cells (APCs/well), transfected with E_52-68-eGFP DNA (squares) or eGFP DNA (triangles), or exogenously pulsed with GST-E_52-68-myc fusion protein (circles) for 24 hours are shown as measured by IL-2 ELISA.

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