Immune pathophysiology in aplastic anemia (AA) is inferred from the clinical successes of immunosuppression. While an autoimmune process is a conceptually attractive hypothesis to explain the destruction of hematopoietic stem cells and resultant cytopenias that cause significant morbidity and mortality, experimental evidence in support of an autoimmune mechanism is only indirect, in part because the identity of the antigens that incite an immune response against target hematopoietic cells remains unknown. A search for hematopoietic antigens targeted in an immune attack in bone marrow failure syndromes may thus provide essential information for understanding the pathophysiology of bone marrow failure and the immune regulation of clonal hematopoietic diseases. One strategy to identify these antigens involves characterization of the proliferation and potential clonal expansion that occurs when T cells recognize an antigen: if destruction of stem cells is induced by specific antigens, an antigen-driven T cell proliferation would be expected. The unique antigen-specific portion of the T cell receptor (TCR), consisting of the most variable portion of the beta chain (VB) called CDR3, can serve as a molecular """"""""signature"""""""" (clonotype) for antigen-specific T cells, and consequently, as a marker of the offending antigen. The goal of this proposal is to define clonotypic """"""""signature"""""""" sequences in specific types of marrow failure that can serve as surrogate markers for pathophysiologic pathways. Ultimately, identification of pathologic T cell clonotypes may help to determine the specific targets of the immune response and aid in the diagnosis of bone marrow failure syndromes. Initially, we will perform analysis of TCR repertoire (VB spectratyping) and determine clonal distribution within expanded VB families. Activated or effector T cells will serve as a source of disease-specific T cells. Next, disease-associated CDR3 sequences derived from oligoclonally skewed VB families will be sequenced, and over-represented, immunodominant clonotypes will be identified. Based on recognition of disease-associated CDR3 sequences, specific molecular probes will be generated and used for testing in blood of patients and controls. To confirm the molecular results, the target cell specificity of potentially pathogenic clonotypes will be tested functionally using a spectrum of hematopoietic target cells. Finally, using probes derived from pathogenic clonotypes, the frequency and patterns of """"""""signature"""""""" TCR clonotypes will be systematically analyzed to characterize utilization patterns and kinetics in distinct subtypes of bone marrow failure. Unlike serologic testing, molecular analysis of the T cell receptor repertoire has not been systematically applied as a diagnostic tool in human diseases. The proposed studies may show that disease-specific clonotypes have diagnostic and prognostic utility, and can provide clues to the nature and role of target antigens in the pathophysioiogy of immune-mediated hematologic diseases but also be applicable to other immune-mediated pathologic conditions.
