P762: hematopoietic progenitor cell-reactive t cell clones can recognize viral epitopes in aplastic anemia bone marrow

Topic: 11. Bone marrow failure syndromes incl. PNH - Biology & Translational Research Background: Aplastic anemia is characterized by bone marrow failure and multi-lineage cytopenia. T cells have been considered key players in immune-mediated aplastic anemia since immunosuppressive therapy with anti-thymocyte-globulin and cyclosporin A is an effective first-line treatment. However, phenotypes and target structures of dominant bone marrow T cell clones are poorly characterized. Aims: We identify immune-phenotypes and target antigens of dominant T cell clones in acquired aplastic anemia bone marrow at the single cell level. Methods: We applied multi-parameter flow cytometry single cell index sorting with subsequent targeted RNA sequencing of T-cell receptor (TCR) αβ and cytokine/transcription factor genes of approx. 8000 T cells from 15 patients with acquired aplastic anemia. Twenty-seven dominant CD8+ T cell clones from eight patients were re-expressed in reporter-T cells to determine reactivity against autologous in vitro expanded hematopoietic progenitor cells and viral epitopes. Results: While none of the 27 re-expressed T cell receptors was activated by CD34- bone marrow, three of them (out of two patients) were activated by magnetically enriched and in vitro expanded CD34+ bone marrow. These progenitor-reactive clones showed a CCR7-CD45RA+CD57+CD28-TBX21+ phenotype, which is indicative of chronic antigen exposure (e.g. viral antigens), and were persistent over time (follow up between five and twenty-four month). Therefore, we tested all 27 TCRs against viral peptide pools and found five of them recognizing epitopes derived from cytomegalovirus (CMV) and one from Epstein-Barr virus (EBV). Interestingly two TCRs recognized both, expanded hematopoietic progenitor cells and viral epitopes. For example, TCR 11A5 was HLA-A*02:01 restricted, recognized expanded progenitor cells, and an epitope derived from the EBV latent membrane protein 1 (LMP1). In vitro testing of an LMP1-minigene did not lead to correct processing/presentation and activation of TCR 11A5 suggesting that additional factors must have contributed to the expansion of T cell clone 11A5 in vivo. Summary/Conclusion: We show for the first time that dominant T cell clones in aplastic anemia bone marrow can be reactive with hematopoietic progenitor cells and epitopes derived from persistent viral infections. Our data suggest a role for chronic viral infections in aplastic anemia pathogenesis as drivers of progenitor cell-directed T cell immune responses. LH and FB contributed equally. Keywords: TCR, Bone marrow failure, Aplastic anemia, Antigen-specific T cells