Antigen-recognition sites of micromanipulated T cells in patients with acquired aplastic anemia

Objective Acquired aplastic anemia (AA) is a rare disorder characterized by pancytopenia and hypocellular bone marrow. Though experimental and clinical data suggest that AA represents a T cell–mediated disease, neither the immune response nor the nature of inciting antigen(s) have been characterized so far. The identification of a restricted T cell repertoire by PCR techniques in total lymphocyte populations supports an antigen-driven T cell response. In order to investigate the clonal composition, we analyzed the gene rearrangements of the T cell receptor (TCR) variable β chain (Vβ) at the single-cell level. Patients and Methods CD3+ T lymphocytes were micromanipulated from peripheral blood and bone marrow samples of 8 AA patients and healthy controls. Subsequently amplified VDJ gene segments of the TCRVβ chain were analyzed for functional rearrangements. More than 500 functionally rearranged TCR loci were studied for Vβ/Jβ gene segment usage and molecular composition of the complementary-determining region 3 (CDR3). Results In comparison to healthy controls, the Vβ sequences confirmed a highly restricted T cell repertoire in AA patients at the single-cell level. Both in bone marrow and peripheral blood a predominance of Vβ13 and Jβ2S7 was observed. Furthermore, individual clonal T-cell expansion was identified in the majority of patients. However, deduced CDR3 amino acid sequences revealed a high variability without common motifs among the 8 patients. Conclusion Individual clonal T-cell expansion with high diversity of the antigen-binding sites among the analyzed patients argues for the predominance of private inciting epitopes in AA.