A critical role for granzyme B, in addition to perforin and TNFalpha, in alloreactive CTL-induced mouse pancreatic beta cell death.
BACKGROUND
The precise effector mechanisms and molecular mediators used by alloreactive cytotoxic T lymphocytes to kill transplanted pancreatic beta cells are poorly defined. We have used mouse (H2b-anti-d) CTLs raised in strains deficient in various key cytotoxic effector molecules to assess the importance of the various signaling pathways mobilized to kill primary mouse pancreatic islet cells, the beta cell line NIT-1, and NIT-1 cells overexpressing dominant-negative FADD and Bcl-2.


METHODS
Death of target cells was assessed using 51Cr release assays.


RESULTS
In short-term assays (<5 hours) beta cell death did not require a functional FasL/Fas pathway, and was not inhibited by Bcl-2. However, the absence of either perforin or granzyme B resulted in cell survival. By contrast, a crucial role for granzyme B was not seen when hematopoietic P815 cells were used as targets, indicating differential regulation of apoptosis. Interestingly, coincubation with CTL for 24 hours revealed an additional but less potent "late phase" of beta cell death that did not require perforin. This delayed death was blocked by dominant-negative FADD, but not by Bcl-2, and was likely to be due to TNFalpha secretion.


CONCLUSIONS
This study suggests that strategies to protect beta cells from allogeneic CTL attack will need to inhibit the perforin/granzyme and probably also the TNFalpha pathway. As there are no known pharmacological approaches to blocking perforin, therapeutic approaches based on overexpressing both dominant negative FADD and an inhibitor of granzyme B may hold promise in prolonging beta cell survival in the allogeneic setting.
Sutton
Vivien R
VR
Cancer Immunology Program, Research Division, Peter MacCallum Cancer Centre, East Melbourne, Australia.
Estella
Eugene
E
Li
Chi
C
Chen
Melanie
M
Thomas
Helen E
HE
Kay
Thomas W
TW
Trapani
Joseph A
JA
D000818:Animals; D016923:Cell Death / Q000276:immunology / Q000502:physiology; D002460:Cell Line; D006084:Graft Rejection; D053804:Granzymes; D066298:In Vitro Techniques; D007515:Islets of Langerhans / Q000276:immunology* / Q000473:pathology*; D016381:Islets of Langerhans Transplantation / Q000276:immunology / Q000473:pathology; D008565:Membrane Proteins / Q000276:immunology*; D051379:Mice; D008810:Mice, Inbred C57BL; D016688:Mice, Inbred NOD; D018345:Mice, Knockout; D052899:Pore Forming Cytotoxic Proteins; D012697:Serine Endopeptidases / Q000276:immunology*; D013602:T-Lymphocytes, Cytotoxic / Q000276:immunology*; D014184:Transplantation, Homologous; D014409:Tumor Necrosis Factor-alpha / Q000276:immunology*
D008565:Membrane Proteins; D052899:Pore Forming Cytotoxic Proteins; D014409:Tumor Necrosis Factor-alpha; C483886:perforin, mouse; D053804:Granzymes; C509214:Gzmb protein, mouse; D012697:Serine Endopeptidases