The CBFA2 (AML1) gene encodes a DNA-binding subunit of the heterodimeric core-binding factor. The CBFA2 gene is disrupted by the (8;21), (3;21), and (12;21) chromosomal translocations associated with leukemias and myelodysplasias in humans. Mice lacking a CBF alpha 2 protein capable of binding DNA die between embryonic days 11.5 and 12.5 due to hemorrhaging in the central nervous system (CNS), at the nerve/CNS interfaces of cranial and spinal nerves, and in somitic/intersomitic regions along the presumptive spinal cord. Hemorrhaging is preceded by symmetric, bilateral necrosis in these regions. Definitive erythropoiesis and myelopoiesis do not occur in Cbfa2-deficient embryos, and disruption of one copy of the Cbfa2 gene significantly reduces the number of progenitors for erythroid and myeloid cells.
Efficient T cell proliferation requires costimulation via CD28/B7 or other pathways. Graft-vs-host disease (GVHD) is caused by activated donor T cells. We have found that the infusion of anti-B7.1 (CD80) + anti-B7.2 (CD86) mAb is effective in eliminating GVHD lethality induced by either CD8+ or CD4+ T cells. Donor CD4+ and CD8+ T cell expansion was inhibited by almost 100-fold as measured by enumerating thoracic duct lymphocytes (TDL) obtained early post-transplant. TDL retained anti-host responsiveness indicating that not all T cells were anergic. Although anti-CD80 or anti-CD86 mAb individually were ineffective in preventing CD8+ T cell GVHD lethality, each mAb was partially effective in CD4+ T cell-mediated GVHD. Because CD80 expression was found to be up-regulated on donor CD4+ TDL post-transplant, the GVHD capacity of donor CD4+ T cells deficient in CD80 was tested and found to be reduced similarly to that seen with anti-CD80 mAb. These studies demonstrate that anti-CD80 + anti-CD86 mAb infusion is effective in preventing GVHD lethality by inhibiting donor CD4+ or CD8+ T cell expansion and provide the first evidence that CD80 expression on donor T cells is critical for optimal GVHD lethality.
The CBFbeta subunit is the non-DNA-binding subunit of the heterodimeric core-binding factor (CBF). CBFbeta associates with DNA-binding CBFalpha subunits and increases their affinity for DNA. Genes encoding the CBFbeta subunit (CBFB) and one of the CBFalpha subunits (CBFA2, otherwise known as AML1) are the most frequent targets of chromosomal translocations in acute leukemias in humans. We and others previously demonstrated that homozygous disruption of the mouse Cbfa2 (AML1) gene results in embryonic lethality at midgestation due to hemorrhaging in the central nervous system and blocks fetal liver hematopoiesis. Here we demonstrate that homozygous mutation of the Cbfb gene results in the same phenotype. Our results demonstrate that the CBFbeta subunit is required for CBFalpha2 function in vivo.
The past year has seen significant advances in our understanding of the role of the B7-CD28/CTLA-4 pathway in T cell activation and self-tolerance. Recent studies have demonstrated that CTLA-4 is a critical negative regulator of T cell activation and autoreactivity, revealing a previously unsuspected means by which costimulation is involved in the maintenance and breakdown of self-tolerance. Manipulation of this costimulatory pathway in animal models of autoimmunity has shown an important role for this pathway in both the initiation and progression of autoimmune diseases.
CasBrE is a neurovirulent murine retrovirus which induces a spongiform myeloencephalopathy in susceptible mice. Genetic mapping studies have indicated that sequences responsible for neurovirulence reside within the env gene. To address the question of direct envelope protein neuroxicity in the central nervous system (CNS), we have generated chimeric mice expressing the CasBrE envelope protein in cells of neuroectodermal origin. Specifically, the multipotent neural progenitor cell line C17.2 was engineered to express the CasBrE env gene as either gp70/p15E (CasE) or gp70 alone (CasES). CasE expression in these cells resulted in complete (>10(5)) interference of superinfection with Friend murine leukemia virus clone FB29, whereas CasES expression resulted in a 1.8-log-unit decrease in FB29 titer. Introduction of these envelope-expressing C17.2 cells into the brains of highly susceptible IRW mice resulted in significant engraftment as integral cytoarchitecturally correct components of the CNS. Despite high-level envelope protein expression from the engrafted cells, no evidence of spongiform neurodegeneration was observed. To examine whether early virus replication events were necessary for pathogenesis, C17.2 cells expressing whole virus were transplanted into mice in which virus replication in the host was specifically restricted by Fv-1 to preintegration events. Again, significant C17.2 cell engraftment and infectious virus expression failed to precipitate spongiform lesions. In contrast, transplantation of virus-expressing C17.2 progenitor cells in the absence of the Fv-1 restriction resulted in extensive spongiform neurodegeneration by 2 weeks postengraftment. Cytological examination indicated that infection had spread beyond the engrafted cells, and in particular to host microglia. Spongiform neuropathology in these animals was directly correlated with CasBrE env expression in microglia rather than expression from neural progenitor cells. These results suggest that the envelope protein of CasBrE is not itself neurotoxic but that virus infectious events beyond binding and fusion in microglia are necessary for the induction of CNS disease.
In mice selectively deficient in CD11b/CD18, a beta 2 integrin, chemoattractant-induced leukocyte adhesion to microvascular endothelium in vivo was reduced. Paradoxically, thioglycollate-induced neutrophil accumulation in the peritoneal cavity was increased and was associated with a significant delay in apoptosis of extravasated cells. The extravasated cells had a near absence of neutrophil phagocytosis and a reduction in oxygen free radical generation, which may contribute to the observed defect in apoptosis. This is supported by our in vitro studies, in which phagocytosis of opsonized particles by human neutrophils rapidly induced apoptosis that could be blocked with CD11b/ CD18 antibodies. Reactive oxygen species are the intracellular link in this process: phagocytosis-induced apoptosis was blocked both in neutrophils treated with the flavoprotein inhibitor diphenylene iodonium and in neutrophils from patients with chronic granulomatous disease, which lack NADPH oxidase. Thus, CD11b/CD18 plays a novel and unsuspected homeostatic role in inflammation by accelerating the programmed elimination of extravasated neutrophils.
Mice homozygous for an Ikaros null mutation display distinct defects in the development of fetal and adult lymphocytes. Fetal T lymphocytes, and fetal and adult B lymphocytes and their earliest progenitors are absent. Postnatally, hematopoietic stem cells give rise to thymocyte precursors that undergo aberrant differentiation into the CD4 lineage and clonal expansion. The lack of NK cells and some gamma delta T cell subsets and a large reduction in thymic dendritic APCs suggest that Ikaros is essential for establishing early branch points in the postnatal T cell pathway. The lymphoid defects detected in Ikaros null mice reveal critical molecular differences between fetal and postnatal hematopoietic progenitors that dictate their ability to give rise to T cells. These studies also establish Ikaros as a tumor suppressor gene acting during thymocyte differentiation. Phenotypic comparison of this null mutation with a severe dominant-negative Ikaros mutation identifies molecular redundancy in the postnatal hemolymphoid system.