The CD3×CD123 DART induces TCR diversification in primary AML samples. Banked PBMCs from AML patients 8 and 9 were grown for 5 days in the presence of CD3×CD123 DART molecules. Purified T cells obtained at baseline (pre-DART) and after DART treatment (post-DART) were evaluated by next-generation sequencing of the TCR-Vα and TCR-Vβ families. Relative levels of 23 specific TCR-Vα (A) and 28 specific TCR-Vβ (B) families are shown here for AML patient 9 and in supplemental Figure 4 for patient 8. These genes were selected for presentation based on being expressed in more than 1% of the T cells in either of the patient samples. (C) TCRβ gene usage. A heat map of the relative frequency of a germline TCR variable (V)-gene allele is plotted relative to the germline joining (J)-gene allele for AML patient 9. The frequency of each V-J combination is represented by the color of the map. (D) D50 index. The D50 index is a quantitative measure of the degree of diversity of T cells within a sample. The D50 is the percentage of T-cell clones that account for the cumulative 50% of the total CDR3s counted in the sample. The more diverse a library, the closer the value will be to 50. Low diversity values are associated with decreased diversity. The data suggest that TCRβ and TCRα diversity increased after DART treatment of both patients.