The TCR diversity of Th1 cells was evaluated in 5 pretreatment AA patients (4 with nonsevere disease and 1 with severe disease) and 4 healthy age-matched donors. (A) A hybrid method was used to purify Th1 cells on the basis of their IFN-γ secretion. In the first step, purified CD4⁺ cells (using negative bead selection) were stimulated in the presence of antibody-coated beads to capture secreted IFN-γ, and the CD4⁺ IFN-γ–secreting cells were enriched by use of magnetic columns (see Methods). The enriched cells were then further sorted by FACS sorter based on positivity for PE-conjugated anti-IFN-γ. The purity of cells was constantly > 95%. Extracted RNA from these cells was used for cDNA synthesis and PCR for 24 Vβ subfamilies (see “Methods”). The spectratypes of Th1 cells were clonal or oligoclonal in the majority of subfamilies. An example of the spectratypes of IFN-γ⁺ and IFN-γ⁻ cells are shown. (B) To calculate the diversity of TCR, CDR3 region, the spectratypes of 24 Vβ subfamilies were scored between 1 and 5, based on the number of peaks. The total value was then calculated and used for comparison between samples. As shown, the CD4⁺ IFN-γ⁺ cells were less diverse (more clonal) than CD4⁺ IFN-γ⁻ cells in AA patients, whereas there was no difference between these types in healthy donors (HD). (C) TCR-β diversity determined by percentages of consensus deep-sequencing clusters in total raw reads without data filtering. In general, there was less diversity in AA patients than in normal healthy donors with regard to spectratyping. The clonal diversity was shown to be statistically significant compared with all deep-sequencing data between the 2 groups (P = .03). (D) Comparison of percentages of dominant TCR-β clones between AA patients and normal healthy donors after removal of sequencing errors and combined clones with the same CDR3 amino acid sequences. Significantly higher percentages of TCR-β dominant clones were seen in AA patients (P values shown), which indicates the clonal expansion of TCRs. (E) CD4⁺ IFN-γ T-cell spectratypes from 2 patients are shown that are either polyclonal (I, II, III, IV), oligoclonal (V), or clonal (VI). Interestingly, in polyclonal spectratypes, large dominant clones (always larger than 25%) were detectable by high-throughput sequencing, which was not the case in healthy donors (C). However, in oligoclonal and clonal spectratypes, the size of the dominant clone was larger in AA patients than in healthy controls (see Figure 6C and supplemental Table 4). Deep sequencing in 2 patients (patient Nos. 6314 and 6235) shown in the figure illustrated immunodominant clones (Vβ7, 9, 2, and 3 in patient 6314 and Vβ1 in patient 6235). The clone size varied from 27% to 81.7% of all reads. Polyclonal spectratypes in AA were clearly associated with immunodominant clones by deep sequencing. Oligoclonal Vβ2 (V) in patient No. 6314 was associated with a clone size of 34.2%, and clonal (VI) spectratype depicted a Vβ3 clone of 81.7%. All dominant clones and the clonal expansions obtained from deep sequencing are shown in the table in panel E. Vβ indicates TCR Vβ chain; CDR3, complementary determining region 3; Jβ, joining region of the TCR-β chain; D region, D region of the TCR-β chain; and UPN, unique patient number.