Overexpression of Fas ligand does not confer immune privilege to a pancreatic beta tumor cell line (betaTC-3).
BACKGROUND
Fas ligand (Fas-L) is thought to provide immune privilege to specific tissues and tumors by inducing an apoptotic signal of cytotoxic T cells expressing its Fas receptor. Purpose. The purpose of this work was to evaluate whether an immortalized insulin-secreting cell line (betaTC-3) gains immune privilege by inducing overexpression of Fas-L.


METHODS
A lipofection technique was used to transfect a betaTC-3 tumor cell line with a plasmid (pcDNA3.1/Zeo) carrying the Fas-L gene and a zeocin resistance gene. Insertion of Fas-L into betaTC was characterized by reverse transcription polymerase chain reaction (RT-PCR) and the ability of transfectants (betaTC-3/Fas-L) to induce apoptosis of Fas-sensitive T cells. Transfectants and control cells were tested for insulin secretion following which 1 x 10(6) insulin-secreting betaTC-3 and betaTC-3/Fas-L cells were subcutaneously implanted into syngeneic, allogeneic, and Fas mutant (lpr) syngeneic mice. Survival of the insulin-secreting cells was then determined by monitoring serum glucose levels in recipients.


RESULTS
Successful transfection of vector resistance gene was achieved in the transfected betaTC-3 cells, which was confirmed by zeocin resistance. RT-PCR in resistant Fas-L clones confirmed the transcription of Fas-L, which was absent in controls. Fas-L transfectants induced 20 +/- 4.2% apoptosis of Fas-sensitive T cells, while controls induced 3.47 +/- 2.3% by flow cytometry (P = 0.04, n = 3). Insulin secretion was equivalent in both betaTC-3 and betaTC-3/Fas-L cells. Syngeneic mice implanted with control betaTC-3 cells died within 3 weeks from hypoglycemia due to overgrowth of betaTC-3 tumor. Implanted Fas-L transfected betaTC-3 cells were killed and had no effect on glycemic status except in Fas mutant hosts, where tumors formed in two of three mice.


CONCLUSIONS
Despite the ability of transfected betaTC-3 cells to induce apoptosis of T cells in vitro, expression of Fas-L provided no immune privilege to these cells in vivo, but paradoxically induced killing of betaTC-3 cells even in syngeneic hosts.
Okamoto
S
S
Department of Surgery, University of Iowa, Iowa City, Iowa, USA.
Takamizawa
S
S
Bishop
W
W
Wen
J
J
Kimura
K
K
Sandler
A
A
D000818:Animals; D017209:Apoptosis / Q000502:physiology; D053222:Fas Ligand Protein; D007340:Insulinoma / Q000276:immunology* / Q000378:metabolism* / Q000503:physiopathology; D008562:Membrane Glycoproteins / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D008807:Mice, Inbred BALB C; D008810:Mice, Inbred C57BL / Q000235:genetics; D008822:Mice, Transgenic / Q000235:genetics; D009368:Neoplasm Transplantation; D010190:Pancreatic Neoplasms / Q000276:immunology* / Q000378:metabolism* / Q000503:physiopathology; D011863:Radioimmunoassay; D013601:T-Lymphocytes / Q000378:metabolism / Q000502:physiology; D014162:Transfection; D014407:Tumor Cells, Cultured; D019014:fas Receptor / Q000235:genetics / Q000378:metabolism
D053222:Fas Ligand Protein; C505658:Fasl protein, mouse; D008562:Membrane Glycoproteins; D019014:fas Receptor