Deep sequencing and flow cytometric characterization of expanded effector memory CD8+CD57+ T cells frequently reveals T-cell receptor Vβ oligoclonality and CDR3 homology in acquired aplastic anemia. Homo sapiens

In this work, we investigated the frequency and oligoclonal expansion of effector CD28-CD57+ memory cells in CD8+ T cell compartments and the T-cell Receptor (TCR) Vβ repertoire in acquired aplastic anemia (AA) to provide additional evidence to the immune hypothesis in AA pathophysiology. Using flow cytometry, deep sequencing, and computational methods, we examined the blood of 32 AA patients and 29 appropriate controls for Vβ usage; eight of these patients showing CD8+CD57+ expansion and three healthy subjects were subjected to TCR deep sequencing to confirm the clonality. We showed that CD8+CD57+ cells were frequently expanded with oligoclonal characteristics in AA, and patients and healthy donors shared CD8+ clonotypes by complementarity-determining region 3 sequences analysis. Overall design: For VDJ combination and CDR3 sequence profiling, DNA was isolated from FACS-sorted CD4+ and CD8+ T cells from nine SAA patients with CD8+CD57+ cell expansion and four healthy subjects (mean, 2.4 μg of DNA; range, 1.2 – 3.4 μg). DNA was also isolated from beads-sorted (Miltenyi Biotec Inc., San Diego, CA) CD8+CD57+ cells from two of the eight SAA patients with enough cells for further analysis (mean, 1.6 μg of DNA; range, 1.0 – 2.3 μg). TCR repertoire sequencing was performed with Illumina HiSeq 2000 sequencer (Illumina, Inc., San Diego, CA).