Examination of the sensitivity of T cells to Fas ligation: induction of allospecific apoptosis.
BACKGROUND
Alloantigen-reactive T cells represent the major barrier to successful organ transplantation. However, it has been shown that cotransplantation of Fas ligand (FasL)-expressing cells can induce functional allograft tolerance in some model systems. In this study, the basis for this tolerance was investigated using a sensitive in vitro assay system.


METHODS
T lymphocytes were activated by coculture with an allogeneic Epstein Barr virus-transformed B-cell line. Samples of the lymphocytes were taken daily and treated with agonistic anti-Fas antibodies or FasL-expressing cells. The time in culture required for development of optimal sensitivity to Fas-mediated apoptosis was assessed by Tdt-mediated nick end labeling (TUNEL) staining and the JAM assay of DNA fragmentation. After the induction of optimal apoptosis, a series of experiments was performed to assess the response of the T-cell population to antigen-specific rechallenge.


RESULTS
Treatment of the allospecific lymphocyte population with anti-Fas antibodies or Fas-L-expressing cells did not induce apoptosis efficiently until between 6 and 7 days after initiation of the mixed lymphocyte culture; this time corresponded with decreases in the ambient interleukin 2 concentration and in Bcl-2 expression. In addition, induction of apoptosis by treatment with the agonistic anti-Fas antibody reduced the lymphoproliferative response of the T-cell population after antigen-specific rechallenge.


CONCLUSIONS
These results give an important indication of the mechanism by which FasL-expressing third-party cells can reduce an allospecific T-cell response by an apoptotic mechanism. Furthermore, they demonstrate that apoptotic tolerance in vivo may only occur after the prolonged period of potentially graft-damaging T-cell activation required for acquisition of sensitivity to Fas-mediated apoptosis.
O'Flaherty
E
E
Department of Surgery, The Medical School, University of Newcastle upon Tyne, United Kingdom.
Ali
S
S
Pettit
S J
SJ
Kirby
J A
JA
D000906:Antibodies / Q000276:immunology; D017209:Apoptosis / Q000276:immunology; D053938:DNA Fragmentation / Q000502:physiology; D004351:Drug Resistance / Q000276:immunology; D000939:Epitopes; D053222:Fas Ligand Protein; D006801:Humans; D020287:In Situ Nick-End Labeling; D007376:Interleukin-2 / Q000378:metabolism; D007519:Isoantigens / Q000276:immunology; D008213:Lymphocyte Activation / Q000502:physiology; D007959:Lymphocyte Culture Test, Mixed; D008562:Membrane Glycoproteins / Q000494:pharmacology*; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism; D013601:T-Lymphocytes / Q000187:drug effects* / Q000276:immunology / Q000502:physiology*; D013997:Time Factors; D014407:Tumor Cells, Cultured; D019014:fas Receptor / Q000276:immunology
D000906:Antibodies; D000939:Epitopes; C505661:FASLG protein, human; D053222:Fas Ligand Protein; D007376:Interleukin-2; D007519:Isoantigens; D008562:Membrane Glycoproteins; D019253:Proto-Oncogene Proteins c-bcl-2; D019014:fas Receptor