T cells use the perforin, but not the FasL, pathway to lyse the 32Dp210 leukemia cell line, even though the leukemic cell lines express Fas and are in vitro sensitive to FasL-induced cell death.

(A) Cytolytic response of splenic T cells that were incubated as effector cells in a cytotoxicity assay with ⁵¹Cr labeled 32Dp210 target cells, as described in “Materials and methods.” Percentages of donor T cells in this experiment were 18% in recipients of B6 T cells (■), 34% in recipients of B6.gld T cells (●), and 26% in recipients of B6.pfp^−/− T cells (▪). B6 bone marrow only, ○. (The effector/target (E/T) ratio was corrected for the percentage of donor splenic T cells in each group. (B) Compared to isotype control (nonfilled overlay curve), FACS staining with anti-Fas antibody (filled curve) demonstrates expression of Fas on the tumor cell lines 32Dp210 and P815 and on the positive control LK35.2, a B-cell hybridoma with known high expression of Fas, but not on the T cell hybridoma cell line DO11.10, which has previously been shown to be Fas negative in its resting state. (C)⁵¹Cr-labeled tumor cell lines 32Dp210 (░) and P815 (▪) and the B-cell hybridoma LK35.2 (with known FasL sensitivity; ■) were coincubated with resting or activated FasL-expressing DO11.10 cells for 8 hours, and cell death was measured by ⁵¹Cr release. Lysis is FasL specific, as shown by the inhibition of cell death by neutralizing FasL antibody, but not by isotype control antibody. (D)⁵¹Cr-labeled tumor cell lines 32Dp210 (░) and P815 (▪) and the B-cell hybridoma LK35.2 (with known FasL sensitivity; ■) were coincubated for 8 hours with L5178Y cells, stably transfected with membrane-bound FasL or with vector alone. Significant lysis as measured by ⁵¹Cr release occurred in the presence of membrane-bound FasL but not with vector-only transfected cells. (E)⁵¹Cr-labeled LPS-stimulated C3FeB6F1/J splenocytes were coincubated for 4 hours with B6, B6.gld, or B6.pfp^−/−splenocytes, which had previously been cocultured for 7 days in the presence of irradiated C3FeB6F1/J splenocytes. Cytolytic activity of allogeneically stimulated splenocytes was measured against⁵¹Cr-labeled LPS blasts.