Fas ligand-mediated lysis of self bystander targets by human papillomavirus-specific CD8+ cytotoxic T lymphocytes - PubMed (original) (raw)

Fas ligand-mediated lysis of self bystander targets by human papillomavirus-specific CD8+ cytotoxic T lymphocytes

M J Smyth et al. J Virol. 1998 Jul.

Abstract

Mouse cytotoxic T lymphocytes (CTL) reactive with a H-2Db-presented 9-mer peptide of the human papillomavirus type 16 protein E7(49-57) (RAHYNIVTF) were generated from the spleen cells of wild-type C57BL/6 (B6) or B6 perforin-deficient (B6.P0) mice. CD8(+) B6 CTL displayed peptide-specific perforin- and Fas-mediated lysis of E7-transfected mouse RMA lymphoma cells (RMA-E7), while CD8(+) CTL from B6.P0 mice lysed RMA-E7 cells via Fas ligand (FasL) exclusively. Rapid and efficient lysis of syngeneic bystander B6 blasts or RMA cells by either B6 or B6.P0 Ag-activated CTL was mediated by a FasL-Fas mechanism. Fas-resistant bystanders were not lysed, nor were allogeneic Fas-sensitive C3H/HeJ (H-2(k)) or BALB/c (H-2(d)) bystander blasts. Interestingly, however, phorbol myristate acetate-ionomycin preactivation of B6.P0 effectors enabled lysis of allogeneic H-2(k) and H-2(d) bystanders even in the absence of antigenic stimulation. Lysis of syngeneic bystander cells was always FasL-Fas dependent and required effector-bystander contact and, in particular, an interaction between CTL LFA-1 and bystander ICAM-1. Thus, in the context of major histocompatibility complex class I molecule-peptide ligation of the T-cell receptors of CD8(+) CTL, neighboring bystander cells that are syngeneic and Fas sensitive and express the adhesion molecule ICAM-1 are potential targets of CTL attack.

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Figures

FIG. 1

FIG. 1

Generation of E749-57-specific B6 and B6.P0 CD8+ CTL. Mice were immunized with E749-57 peptide, and responding CTL were induced as described in Materials and Methods. The resultant B6 (A) and B6.P0 (B) CTL cultures were assayed for specific cytolytic activity toward RMA-E7 cells, RMA-S cells alone, or RMA-S cells pulsed with the E749-57 peptide or the OVA257-264 peptide in a 4-h 51Cr release assay. Some effectors were pretreated with anti-CD8 MAb and complement or anti-CD4 MAb and complement prior to examination of the cytolysis of RMA-E7 target cells. Direct cytotoxicity was assessed at the four effector/target ratios illustrated (104 target cells/well). Cytotoxicity was expressed as specific 51Cr release after subtraction of spontaneous 51Cr release, which was less than 15%. These results were calculated as the means ± standard errors of duplicate samples and are representative of three experiments performed.

FIG. 2

FIG. 2

B6 and B6.P0 CD8+ CTL specific for E749-57 mediate bystander lysis. (A) Bystander lysis by B6 (solid symbols) and B6.P0 (open symbols) CD8+ CTL specific for E749-57 was examined at the four effector/target ratios illustrated (104 51Cr-labeled bystanders and 104 cold RMA-E7 cells/well) against 51Cr-B6 blasts and RMA-E7 cells (squares), 51Cr-EL4 and RMA-E7 cells (circles), or 51Cr-RMA and RMA-E7 cells (triangles) in a 4-h 51Cr release assay. Bystander lysis of B6 blasts or EL4 cells was not observed in the absence of RMA-E7 cells or presence of RMA cells (<10% lysis at all effector/target ratios). (B) Direct lysis by B6 (solid symbols) and B6.P0 (open symbols) CD8+ CTL specific for E749-57 was examined at the four effector/target ratios illustrated (104 51Cr-labeled RMA-E7 target cells and 104 cold bystanders/well) against 51Cr-RMA-E7 cells and B6 blasts (squares), 51Cr-RMA-E7 and EL4 cells (circles), or 51Cr-RMA-E7 and RMA cells (triangles) in a 4-h 51Cr release assay. Cytotoxicity was expressed as specific 51Cr release after subtraction of spontaneous 51Cr release, which was <15%. These results were calculated as the means ± standard errors of duplicate samples and are representative of two experiments performed.

FIG. 3

FIG. 3

Bystander lysis by E749-57-specific B6 or B6.P0 CD8+ CTL is FasL mediated. Direct lysis by B6 (A) and B6.P0 (B) CTL specific for E749-57 (at the four effector/target ratios illustrated; 104 51Cr-labeled RMA-E7 target cells and 104 cold B6 blasts/well) and bystander lysis by B6 (C) and B6.P0 (D) CTL specific for E749-57 (at the four effector/target ratios illustrated; 104 51Cr-labeled B6 blasts and 104 cold RMA-E7 cells/well) were examined in a 4-h 51Cr release assay. These assays were performed in the absence (solid squares) or presence of Fas-Fc (open circles), EGTA (solid circles), Fas-Fc and EGTA (solid triangles), or TNFR-Fc (open squares). Cytotoxicity was expressed as specific 51Cr release after subtraction of spontaneous 51Cr release, which was <15%. These results were calculated as the means ± standard errors of duplicate samples and are representative of two experiments performed.

FIG. 4

FIG. 4

Mode of CTL activation determines type and level of bystander lysis. Blasts of B6 (H-2b), B6.β2μ0 (H-2b), C3H/HeJ (H-2k), or BALB/c (H-2d) origin or EL4 cells (H-2b) were 51Cr labeled and used as targets for B6.P0 anti-E749-57 CTL added to Ag-bearing RMA-E7 cells (1:1 ratio) (A), B6.P0 anti-E749-57 CTL prestimulated with PMA-ION (for 3 h) and then added to RMA-E7 cells (1:1 ratio) (B), B6.P0 anti-E749-57 CTL alone (C), and B6.P0 anti-E749-57 CTL prestimulated with PMA-ION for 3 h (D). Lysis by B6.P0 anti-E749-57 CTL was examined in a 4-h 51Cr release assay at the four effector/target ratios illustrated. (E) Lysis of blasts and EL4 target cells by doubling dilutions of srFasL. Cytolysis was expressed as specific 51Cr release after subtraction of spontaneous 51Cr release, which was <15%. These results were calculated as the means ± standard errors of duplicate samples and are representative of two experiments performed.

FIG. 5

FIG. 5

No FasL-mediated activity in culture supernatants from activated CTL. Anti-E749-57 CTL from B6 and B6.P0 mice (effectors) were stimulated with PMA-ION or incubated with Ag-bearing RMA-E7 cells (stimulators) as for a 4-h assay (effector/target ratio of 50:1). Supernatants were collected at 4 h and evaluated for their ability to lyse 51Cr-labeled B6 blasts, RMA cells (Fas sensitive), or EL4 cells (Fas insensitive) in a 4-h 51Cr release assay. srFasL (sFasL) was used as a positive control. Cytolysis was expressed as specific 51Cr release after subtraction of spontaneous 51Cr release, which was <15%. These results were calculated as the means ± standard errors of duplicate samples.

FIG. 6

FIG. 6

CTL LFA-1–bystander ICAM-1 interaction is critical for bystander lysis. (A) Direct lysis. B6.P0 anti-E749-57 CTL and 51Cr-labeled RMA-E7 target cells were coincubated for 4 h (open square). In some tests MAb (anti-LFA-1 [20 μg/ml; open circles], anti-CD8 [20 μg/ml; solid circles], or anti-Thy 1.1 [20 μg/ml; solid triangles]) was added 1 h after the coincubation commenced. (B) Bystander lysis. B6.P0 anti-E749-57 CTL and 51Cr-labeled B6 bystanders were incubated alone (open squares) or together with RMA-E7 target cells in the absence (solid squares) or presence of MAb (symbols as in panel A) for 4 h. (C) Bystander lysis. B6.P0 anti-E749-57 CTL and RMA-E7 stimulator cells were coincubated for 1 h prior to the addition of 51Cr-labeled B6 blasts and MAbs as in panels A and B for a further 4 h. (D) B6.P0 anti-E749-57 CTL and RMA-E7 stimulator cells (squares) or d12S effector cells (circles) were incubated for 4 h with 51Cr-labeled B6 blasts (solid symbols) or B6.ICAM-10 blasts (open symbols). Lysis was determined at the four effector/target ratios illustrated, as described in the legends to Fig. 1 and 2, and results were calculated as the means ± standard errors of duplicate samples.

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