Fas- and Fas Ligand Rather Than Perforin-Mediated Apoptosis Has a Dominant Role in Target Cell Death in Murine Models of Immune- Mediated Bone Marrow Failure
Authors: Chen Jichun , Omokaro Stephanie O. , Sarcon Annahita K. , Young Neal 
Published: April 2020

Abstract
Fas-Fas ligand and perforin-granzyme are two important cell death pathways associated with cytotoxic T cell induced target cell apoptosis. In patients with immune-mediated aplastic anemia, the development of bone marrow (BM) failure is associated with up-regulation in Fas ligand expression on effector cytotoxic T cells and elevated Fas expression on target BM cells. In some aplastic anemia patients, peripheral blood T lymphocytes also carry polymorphisms in the perforin gene which have been associated with familial hemophagocytosis. These findings suggested that Fas ligand/Fas might be the key signaling molecules mediating cell destruction while perforin might also play a role in the development of BM failure in patients with aplastic anemia. We have modeled immune-mediated BM failure in the mouse by infusing allogeneic lymph node (LN) cells from C57BL/6 (B6) donors into sublethally-irradiated CByB6F1 and C.B10 recipients that are mismatched at either major histocompatibility (MHC) or minor histocompatibility (minor-H) loci. Expansion and activation of allogeneic T cells results in increased production of the inflammatory cytokines gamma interferon and tissue necrosis factor alpha in recipient BM, massive BM cell destruction, severe marrow hypoplasia, and fatal pancytopenia. In the current study, we directly tested the roles of Fas, Fas ligand and perforin in the development of BM failure by using murine models with spontaneous mutations at the lymphoproliferation (lpr) and generalized lymphoproliferative disease (gld) loci, or with germline deletion of the gene perforin (prf−/−). Fas and Fas ligand-deficient lpr and gld mutant mice had no evidence of hematopoietic deficiency despite their autoimmune environment and marked lymphoproliferation. LN cells from lpr and gld mice caused significantly less apoptosis to minor-H mismatched C.B10 BM cells when co-cultured in a cytotoxicity assay in vitro, in comparison to LN cells from wild-type B6 mice. Infusion of lpr, gld, and B6 donor LN cells into sub-lethally irradiated CB10 recipients all caused massive T cell expansion in recipient BM with high level expression of CD11a, indicative of T cell activation, but only B6 LN cells caused severe BM destruction. In contrast, recipients of lpr and gld LN cells had only mild to moderate pancytopenia and marrow hypocellularity. We inferred from these results that disruption of the Fas ligand/Fas signaling pathway effectively abrogated immune mediated marrow destruction. To test the role of perforin in BM failure, we first analyzed prf−/−- mice and found no obvious change in cellular composition in lymphohematopoietic tissues in comparison to wild-type B6 controls. LN cells from prf−/− mice showed reduced ability to induce C.B10 BM cell apoptosis in an in vitro cytotoxicity assay when compared to wild-type B6 LN cells. Infusion of 5–10 million prf−/− LN cells into CByB6F1 and C.B10 recipients produced obvious BM failure in both recipient types with pancytopenia and marrow hypoplasia about 80–90% as severe as in control recipients of 5 million B6 LN cells. In both CByB6F1 and C.B10 recipients, infused prf−/− LN cells resulted in less T cell expansion, a similar level of T cell activation, higher proportions of T cells containing gamma-interferon and tissue necrosis factor-alpha, and a higher proportion of T cells expressing the Fas ligand CD178, in comparison to the infused B6 LN cells. We conclude that Fas-Fas ligand-mediated transmembrane signaling provides the major cell death pathway, while perforin-granzyme-mediated exocytosis plays a minor role, in BM cell destruction in animal models of immune-mediated BM failure.