Aplastic anemia (AA) and other types of bone marrow failure have clinical and laboratory features consistent with an autoimmune pathophysiology, with a diversity of inciting antigens, including viruses, chemicals, and drugs. Whatever its specific etiology, a majority of patients respond with hematologic improvement after immunosuppressive therapies. One important clinical feature of AA is its evolution, sometimes years after normalization of blood counts, to other hematologic diseases such as paroxysmal nocturnal hemoglobinuria (PNH), which derive from clones of hematopoietic stem cells. In the clinic, we have employed a regimen that incorporates mycophenolate mofetil, as well as delayed addition of cyclosporine, to standard antithymocyte globulin, in an effort to induce tolerance in patients with severe aplastic anemia on presentation. With accrual almost complete, hematologic response rates at 3 and 6 months are comparable to those achieved by standard treatment; relapse, evolution, and survival analyses will require longer periods of evaluation. In the laboratory, efforts have concentrated on the incitement of aplastic anemia by an unknown virus (see Z01 HL 02319-14 HB), the aberrant immune response, and the problem of clonal evolution to other hematologic diseases. In efforts to more specifically characterize the immune response, we have utilized the methods of flow cytometry and spectratyping to determine expansion of V-beta TCR families of T cells and skewing of CDR3 expression within expanded families, as indicators of antigen-driven clonal T cell proliferation. Expecially within the CD8 cytotoxic lymphocyte population, we found that a large proportion of aplastic anemia and PNH patients show oligoclonal T cell expansion, and we have identified individual CDR3 sequences within these families. Molecular measurment of CDR3 correlates with disease status pre- and post-immunosuppression, but the patterns differ depending on whether ATG or cyclosphosphamide is used. CDR3 clonotypes are sought as markers of the immune response in patients of similar HLA background. Ultimately, CDR3 regions of interest may be incorporated into recombinant human TCR genes and in turn transduced into immortalized effector cells, for determiantion of antigen specificity. We also have employed DNA chip analysis to examine CD34 hematopoietic cells, both normal and from patients with marrow failure syndromes. Limited numbers of CD34 cells from aplastic anemia cases have been pooled and cRNA pre-amplified. For this disease, target cells show evidence an enhanced genetic program of expression of genes related to immune system activation, apoptosis, and stress, and decreased expression of proliferation genes. While paired cell lines of normal and PNH phenotype show little overall difference in transcriptomes, paired primary CD34 cells from individual patients show marked upregulation of immune and apoptosis genes in the abnormal putative PIG-A-negative population; these results are consistent with our previous cell culture data using similar cell populations. In myelodysplasia, specifically up- and down-regulated genes have been observed for CD34 cells from patients with well-defined cytogenetic abnormalities in myelodysplasia . Trisomy 8, but not monosomy 7, also is associated with immune abnormalities related to hematopoiesis, as cytogenetically abnormal cells are more likely than normal cells to express Fas and to be apoptotic.
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