the mouse fasl locus and known restriction sites were used to construct the targeting vector and diagnose homologous recombination in embryonic stem ( es ) cells and gene - targeted ( fasl , fasl ) mice .
targeting knock - in vectors were made with the loxp / pgkneo / loxp cassette cloned into the pac1 site . targeting constructs for the mutant fasl mice ( supplementary fig .
all experiments with mice were performed according to the guidelines of the animal ethics committees of our institutions .
mice were killed at 6 , 12 or 20 weeks for analysis and further cohorts were monitored daily for morbidity and killed when showing signs of illness .
tissues were fixed for microscopic analysis in 80% histochoice ( amresco)/20% ethanol or 10% buffered formalin and embedded in paraffin and conventional histopathology was performed on hematoxylin plus eosin stained sections . for detailed methods for immunohistochemical staining , immunofluorescent staining , confocal microscopy , cell preparation , flow cytometric analysis , elisa , aicd , chromatography , viral infection and target cell killing , t cell proliferation assays and western blotting refer to the online methods version .
statistical analysis was performed using the student 's t - test , log rank ( mantel - cox ) test for survival curves or one - way analysis of variance using turkey 's comparison test to compare multiple groups where appropriate .
the mouse fasl locus and known restriction sites were used to construct the targeting vector and diagnose homologous recombination in embryonic stem ( es ) cells and gene - targeted ( fasl , fasl ) mice .
targeting knock - in vectors were made with the loxp / pgkneo / loxp cassette cloned into the pac1 site . targeting constructs for the mutant fasl mice ( supplementary fig .
all experiments with mice were performed according to the guidelines of the animal ethics committees of our institutions .
mice were killed at 6 , 12 or 20 weeks for analysis and further cohorts were monitored daily for morbidity and killed when showing signs of illness .
tissues were fixed for microscopic analysis in 80% histochoice ( amresco)/20% ethanol or 10% buffered formalin and embedded in paraffin and conventional histopathology was performed on hematoxylin plus eosin stained sections . for detailed methods for immunohistochemical staining , immunofluorescent staining , confocal microscopy , cell preparation , flow cytometric analysis , elisa , aicd , chromatography , viral infection and target cell killing , t cell proliferation assays and western blotting refer to the online methods version .
statistical analysis was performed using the student 's t - test , log rank ( mantel - cox ) test for survival curves or one - way analysis of variance using turkey 's comparison test to compare multiple groups where appropriate .
 fas ligand ( fasl ) , an apoptosis - inducing member of the tnf cytokine family and its receptor , fas , are critical for shutdown of chronic immune responses1 - 3 and prevention of autoimmunity4,5 . 
 accordingly , mutations in their genes cause severe lymphadenopathy and autoimmune disease in mice6,7 and humans8,9 . fasl function is regulated by deposition in the plasma membrane and metalloprotease - mediated shedding10,11 . 
 we generated gene - targeted mice that selectively lack either secreted fasl ( sfasl ) or membrane - bound fasl ( mfasl ) to resolve which of these forms is required for cell killing and to explore their hypothetical non - apoptotic activities . 
 mice lacking sfasl ( fasls/s ) appeared normal and their t cells readily killed target cells , whereas t cells lacking mfasl ( faslm/m ) could not kill cells through fas activation . 
 faslm/m mice developed lymphadenopathy and hyper - gammaglobulinaemia , similar to faslgld / gld mice , which express a mutant form of fasl that can not bind fas , but surprisingly , ( on a c57bl/6 background ) faslm/m mice succumbed to sle - like autoimmune kidney destruction and histiocytic sarcoma , diseases that occur only rarely and considerably later in faslgld / gld mice . 
 these results demonstrate that mfasl is essential for cytotoxic activity and constitutes the guardian against lymphadenopathy , autoimmunity and cancer whereas excess sfasl appears to promote autoimmunity and tumorigenesis through non - apoptotic activities . 
Methods Summary
Generation of FasL mutant mice
Analysis of FasL mutant mice
Supplementary Material