Utlilizing Mutant Mice To Dissect Immune Bone Marrow Failure: Markedly Reduced Cytotoxicity of Lymphocytes from Fas- and Fas Ligand-Deficient Mice in Lymph Node Cell-Mediated Aplastic Anemia.

Key cellular events in the development of aplastic anemia are activation and expansion of T cells that are destructive of hematopoietic tissue. Human immune-mediated bone marrow failure has been modeled in mice by infusion of lymph node (LN) cells into major- or minor-histocompatibility antigen mismatched recipients; oligoclonal T cell expansion causes indiscriminant hematopoietic cell destruction, marked marrow hypoplasia, and fatal pancytopenia. We have utilized this simple model in order to dissect the molecular events responsible for T cell mediated, selective organ destruction. In mice with spontaneous mutations: lymphoproliferation (lpr) and generalized lymphoproliferative disease (gld), animals have an abnormal increase in lymphocytes and develop clinical manifestations similar to those seen in patients with systemic lupus erythematosus (SLE). In contrast to human SLE, the lpr and gld bone marrow appear unaffected by this typically self-destructive process. We examined C57BL/6 (B6) mice that carry the lpr and gld mutations along with normal B6 mice to define cellular and functional differences as they relate to BM damage. In our initial analyses of peripheral blood (PB), we found a reduced CD4 T cell proportion in lpr and gld mice (5.5±1.5%, 4.3±0.6%, 9.9±0.9%) and a reduced CD8 T cell proportion in gld mice when compared to B6 controls (13.4±1.9%, 7.9±0.9%, 12.7±1.8%). In the BM, both CD4 (1.5±0.3%, 2.3±0.3%, 1.0±0.1%) and CD8 (2.9±0.3%, 3.1±0.7%, 2.6±0.2%) T cell proportions were slightly increased in lpr and gld mice, while total number of BM cells was not significantly different among the three genotypes (438±48, 324±18, 350±27, millions). These findings indicated no PB lymphocyte accumulation and no BM destruction in lpr or gld mice. We then tested the hypothesis that lpr and gld BM cells were more resistant to immune attack by infusing 5 x 10⁶ lymph node (LN) cells from minor-histocompatibility antigen-mismatched C.B10 mice into sublethally-irradiated (5 Gys) lpr, gld, and B6 recipients. At two weeks, all recipients infused with CB10 LN cells had reduced blood neutrophils, and gld and B6 recipients also had reduced RBCs, as compared to mice that did not receive LN cell infusions. This result suggests that lpr and gld hematopoietic cells, like those from B6 mice, are susceptible to immune-mediated destruction. We then examined lymphocyte function by co-incubating lpr, gld and B6 LN cells (effectors) with C.B10 BM cells (targets) in a cytotoxilux assay in vitro. B6 effectors produced 51–78% more apoptotic targets than did gld and lpr effectors. This difference was confirmed by the infusion of lpr, gld (15 x 10⁶ cells/recipient) and B6 (5 x 10⁶ cells/recipient) LN cells into sub-lethally irradiated CB10 mice in vivo. At three weeks, enumeration of blood neutrophils (0.69±0.60, 1.27±0.83, 0.08±0.03), RBCs (3.58±0.42, 4.80±1.20, 4.76±0.36), platelets (274± 94, 400±156, 298±91) and total BM cells (87± 23, 195±78, 82±6, millions) demonstrated that, despite massive lymphoproliferation, lymphocytes from lpr and gld mice were less effective in mediating BM cell destruction. Our results provide evidence supporting the role of Fas and Fas-ligand mediated programmed cell death in immune-mediated BM destruction. The ready availability of genetically modified mice with well characterized functional defects should allow a full description of the molecular pathways in immune-mediated marrow failure in this model.