Clonal expansion and immune phenotypes of bone marrow T cells in aplastic anemia. (A) Bone marrow single-cell TCRαβ sequencing and immune phenotyping of patient 230 as an example. Single cells are arranged in columns with each column representing 1 single cell. The top bar indicates TCR sequences; adjacent cells with the same color in the top bar share identical TCRαβ CDR3 amino acid sequences. Clonal expansion was defined as the detection of at least 2 cells with identical TCRαβ CDR3 sequences. The upper part of the heat map indicates single-cell gene expression determined by targeted panel sequencing. The lower part of the heat map visualizes corresponding flow cytometry index-sort data. (B) Numbers of expanded bone marrow T-cell clones per patient. (C) Expression of CD4 and CD8 on expanded T-cell clones. Data points indicate mean frequencies of CD4⁺ or CD8⁺ expanded T-cell clones per patient. (D) Frequencies of expanded T-cell clones within total CD8⁺ T cells. (E) Immune phenotype of index-sorted T cells from patient 230 as an example. Numbers indicate percentages within quadrants. (F) Phenotype determined by flow cytometry (left and middle panels) and RNA sequencing (right panel) for all n = 15 patients. Each data point represents the frequency of positive clones within all expanded (red) or nonexpanded (gray) CD8⁺ clones of each patient. Boxes reach from the first to third quartiles, lines within boxes indicate medians. The upper whisker extends from the hinge to the largest value, no further than 1.5× interquartile range (IQR). The lower whisker extends from the hinge to the smallest value, at most 1.5× IQR. Statistics were calculated using Wilcoxon signed-rank test and adjusted for multiple testing by Bonferroni correction. ∗P < .05, ∗∗P < .01, ∗∗∗P < .001, and ∗∗∗∗P < .0001. CM, central memory; EM, effector memory; EMRA, effector memory with CD45RA expression.