Differential Role of Fas/Fas Ligand Interactions in Cytolysis of Primary and Metastatic Colon Carcinoma Cell Lines by Human Antigen-Specific CD8⁺ CTL

Fas-dependent and Fas-independent pathways in CTL-mediated lysis of IFN-γ-pretreated SW480 and SW620 cells. A, CTL-mediated lysis of IFN-γ-pretreated SW480 or SW620 cells was measured by ⁵¹Cr release at multiple E:T ratios (in a 12-h assay; spontaneous release of ⁵¹Cr for all targets was ≤13%). Assays were also conducted in the absence and presence of anti-Fas mAb, clone ZB4 (10 μg/ml). Because of the size or overlap of symbols, error bars may be partly masked. Percent specific lysis of untreated SW480 cells at E:T ratios of 20:1 and 5:1, 8 ± 4 and 4 ± 1; for untreated SW620 cells, 22 ± 3 and 13 ± 1. B, IFN-γ-pretreated SW480 or SW620 cells were radiolabeled with [¹²⁵I]IUdR and tested against anti-ras CTL (E:T ratio, 10:1; 12 h assay) to determine the extent of nuclear damage. Assays were also conducted in the absence and presence of Ab, as in A. Data in A and B were expressed as the mean ± SEM of triplicate cultures and are representative of five and four separate experiments, respectively. C, Effect of IFN-γ pretreatment on expression of the mutant ras Val12 determinant in SW480 and SW620 cells. Cell lysates were collected from tumor cells, as shown, and then assayed in titrating amounts in a mutant ras Val12-specific ELISA. Data were expressed as the mean OD (490 nm) ± SEM of five separate experiments.

Comparison of SW480 to SW620 cells for functional Fas expression. A, Tumor cells, either untreated (−IFN-γ) or pretreated with IFN-γ (+IFN-γ), were incubated with CH-11 or an isotype-matched Ab (at the highest concentration tested, 3 μg/ml). Cell death was measured by ⁵¹Cr release (18 h assay). B, In parallel, these same tumor cell lines were incubated with sFasL or TNF-α (at the highest dose tested, 100 ng/ml; ∼1000 U/ml). The extent of CH-11 (A)- or sFasL (B)-induced lysis of SW620 cells was the same with or without IFN-γ pretreatment. Also, in A and B, Jurkat cells served both as a Fas- and TNF-sensitive positive control. C, Anti-MART-1_27–35-specific CTL were incubated either in the absence (“unstimulated”) or presence (“pre-activated”) of immobilized anti-CD3 mAb. Afterward, one aliquot was analyzed for FasL expression by flow cytometry, while a second aliquot was used as effectors in ⁵¹Cr release assays against the indicated target cells (E:T ratio, 10:1). CH-11 or isotype (1μ/ml) used as in A. D, In additional control experiments, clone ZB4 was tested at multiple concentrations for potency, based on its ability to inhibit CH-11 (1 μg/ml)-mediated lysis of Jurkat and IFN-γ-pretreated SW480 cells. Percent specific lysis of Jurkat and IFN-γ-pretreated SW480 cells with CH-11, but without ZB4, 94 ± 5 and 48 ± 2, respectively. Data in all panels were expressed as the mean ± SEM of triplicate cultures and are representative of four separate experiments for A–C and two separate experiments for D.

Apoptotic cell death induced by agonistic anti-Fas mAb. IFN-γ-pretreated SW480 (A) or SW620 (B) cells were incubated without (dashed line) or with (solid thick line) CH-11 (1 μg/ml). Solid thin line represents untreated tumor cells incubated with CH-11. (C) Jurkat cells were incubated without (solid thin line) or with CH-11 (solid thick line). After a 24-h incubation, all tumor cell populations were analyzed for apoptotic death by the TUNEL assay. Dotted line in all panels denotes tumor cells (Jurkat or IFN-γ-pretreated SW480 or SW620 cells) cultured with CH-11 but analyzed by the TUNEL assay without the enzyme. Data are representative of four separate experiments.

Ag-specific CTL-mediated lysis of IFN-γ-pretreated SW480 and SW620 cells involves distinct effector mechanisms. A, Cytotoxicity against IFN-γ-pretreated tumor cells (E:T ratio, 10:1) was assayed in the presence of the indicated blocking mAb. Isotype-matched Ab had no significant inhibitory effect (not shown). Assays were also conducted in the presence of CMA (10 μM). B, In a separate experiment, cytotoxicity against IFN-γ-pretreated tumor cells was assayed in the presence of anti-FasL mAb, anti-Fas mAb, CMA, or a combination of both anti-Fas mAb and CMA. Ab, 10 μg/ml; CMA (10 μM). Data in A and B were expressed as the mean ± SEM of triplicate cultures and are representative of four separate experiments. CMA (at an optimal inhibitory dose of 10 μM, based on titration experiments) did not alter viability of either cell population, as determined by trypan blue dye exclusion and spontaneous release of ⁵¹Cr from radiolabeled targets. The inhibitory effect was at the level of the effector cell, because pretreatment of the CTL, but not of the target cells, resulted in reduction of lysis (not shown).

IFN-γ-pretreated SW480 and SW620 cells were lysed by a second, independently isolated, HLA-A2-restricted, anti-ras Val12-specific CD8⁺ CTL line. Using a second anti-ras Val12-specific CD8⁺ CTL line, the nature and spectrum of cytotoxic mechanisms used against Ag-bearing, IFN-γ-pretreated SW480 and SW620 cells was determined as in Figs. 2 and 5. ⁵¹Cr release assays were conducted in the absence (“no Ab”) and presence of ZB4 or an isotype control, or CMA to determine the role of Fas-dependent and Fas-independent pathways in Ag-specific tumor cell killing of SW480 (A) or SW620 (B) tumor cells. Data in both panels were expressed as the mean ± SEM of triplicate cultures and are representative of four separate experiments.

Induction of FasL expression on anti-ras Val12 CD8⁺ CTL lines after TCR stimulation. Anti-ras 4–12(Val12)-specific CTL and anti-ras 5–14(Val12)-specific CTL were stimulated in vitro with immobilized anti-CD3 mAb or adherent IFN-γ-pretreated SW480 or SW620 cells. Parallel CTL cultures incubated in the absence of anti-CD3 mAb or tumor cells were characterized as unstimulated. Nonadherent cell suspensions were then analyzed for cell-surface expression of both CD8 and FasL markers. Data are representative of three separate experiments.

Effect of 5-FU treatment on susceptibility of SW480 and SW620 cells to Fas-mediated lysis by agonistic anti-Fas mAb. SW480 and SW620 cells were pretreated with 5-FU at multiple concentrations and recultured with CH-11 or an isotype control Ab (MOPC-104E) (each at 1 μg/ml). Untreated (−IFN-γ) and IFN-γ-pretreated tumor cells (in the absence of 5-FU pretreatment) were examined at the same time for comparison (see insert). Cell death was determined by ⁵¹Cr release assays (18 h assay). 5-FU pretreatment alone, at the concentrations shown, did not alter SW480 or SW620 cellular viability based on trypan blue dye exclusion and spontaneous release of ⁵¹Cr relative to the untreated controls. Data were expressed as the mean ± SEM of triplicate cultures and are representative of four separate experiments.

5-FU sensitizes SW480, but not SW620, to Fas-mediated cytotoxicity in response to Ag-specific CTL. A, SW480 cells were pretreated with IFN-γ or 5-FU as in Fig. 8. After treatment, tumor cells were used as targets in ⁵¹Cr release assays, and lytic activity was determined using anti-ras 4–12(Val12)-specific CTL at multiple E:T cell ratios. B, In a separate experiment, the nature of the CTL effector mechanisms against 5-FU-pretreated (30 μg/ml) SW480 cells was determined (E:T ratio, 10:1) in the absence (“none”) and presence of ZB4 or an isotype control (MOPC-21) (each at 10 μg/ml) or CMA (10 μM). C, Same as in A, except targets were SW620 tumor cells. Data in all panels were expressed as the mean ± SEM of triplicate cultures and are representative of four separate experiments.