Phenotypic and functional properties of killer DC-DC hybrids.

(A) The indicated cell lines were examined for CD95L and β-actin mRNA expression by RT-PCR. cDNA encoding CD95L or β-actin was amplified to serve as a positive control. The data are representative of 3 independent experiments. (B) The indicated cell lines were stained with anti-CD95L mAb (filled histograms) or isotype-matched control mAb (open histograms). CD95L-transfected and nontransfected L5178Y cell lines were stained in parallel to serve as positive and negative controls, respectively. (C) Two parental DC preparations, the XS106-CD95L clone (A/J origin) and splenic DCs (BALB/c origin), and the resulting killer DC-DC hybrid were stained with the indicated haplotype-specific mAbs (filled histograms) or isotype-matched control IgG (open histograms). Modest cross-reactivity of anti–H-2K^d mAb (SF1-1.1) with the H-2K^k determinant (top left) has been reported previously.26 (D) Killer DC-DC hybrids (clone 8) were stained with mAbs against the indicated surface molecules (filled histogram) or control IgG (open histogram). (E) The killer DC-DC hybrid line (closed triangles) and clone 8 (closed circle), as well as the control DC-DC hybrid line (closed squares), were examined for their capacity to kill Jurkat targets in a standard 18-hour³H-release assay at the indicated E/T ratios. Data are representative of 3 independent experiments, showing the means ± SD of percentage specific lysis from triplicate samples. (F) The killer DC-DC hybrid clone 8 was tested for cytotoxicity against the Jurkat target at the E/T ratio of 1.0 in the presence of neutralizing mAb against CD95L (Kay-10) or control IgG. The data are representative of 2 independent experiments, showing the means ± SD (n = 3). (*P < .01; brackets indicate groups being compared.)