B7.1-expressing cells can downregulate CD3-triggered cell death (A), DNA fragmentation (B), FasL mRNA induction (C), and BclXL upregulation (D). (A) DWT6.11 cells were seeded for 12 hours in a 24-well plate in the presence of MoAbs or in the presence of either L cells (L) or L cells expressing a transfected B7.1 cDNA (LB7.1), as indicated, with a 3:1 (DWT6.11:L) cell ratio. After this incubation period cells were collected and PI stained followed by cytofluorometry as described in the Materials and Methods. PI staining versus the number of nuclei is shown. Numbers above histograms indicate the percentage of apoptotic nuclei. Where indicated, the CTLA4Ig recombinant protein was added 15 minutes before treatment. (B) DNA was extracted from cells collected at 8 hours of incubation and analyzed on a 1% agarose gel containing ethidium bromide. (C) mRNA was extracted from cells collected at 6 hours of incubation, followed by reverse transcription and PCR using FasL, IL-2, or β2 microglobulin primer pairs as described in the Materials and Methods. Amplification products were analyzed on a 1% agarose gel, followed by ethidium bromide staining. (D) Proteins were extracted followed by SDS-PAGE fractionation and Western blotting with the indicated polyclonal serum. Results are representative of at least three independent experiments.
