Figure 3.
SPI-6 is expressed in primary DCs and prevents cytotoxicity. Primary bone marrow DCs were either left untreated or treated for 48 h with Lz-CD40L and tested for (a) B7-2 expression by FACS® analysis, (b) SPI-6 expression using Western blot analysis, and (c) CTL sensitivity as described in Fig. 1 a. (d) Spleen cell DCs were isolated by FACS® sorting using CD11c and B7-2 expression as markers. Cells were isolated either from untreated mice (sorted for immature DC, B7-low) or from FGK45-injected mice (sorted for mature DCs, B7-high) and SPI-6 as well as GAPDH expression was determined directly by rtPCR. (e) Primary human monocyte–derived DCs were treated with Lz-CD40L or LPS for 24 h and tested for PI-9 expression (i.e., the human homologue of SPI-6) by Western blot analysis. In all cases lysates from equal numbers of cells were applied on SDS-PAGE (0.5 × 106 cells) and equal protein loading was further checked by ponceau S staining and control Western for MAPK (bottom). (f) D1 cells transduced with control retrovirus (white squares) or SPI-6-encoding retrovirus (black squares) were tested for SPI-6 expression by Western blot (inset) and CTL sensitivity by DNA fragmentation.