Figure 6.

TCR repertoire distribution of CD8⁺ T cells based on PD-1 expression in CR and relapsed AML patients. (A) Expression of PD-1 was determined by flow cytometry. Samples from 5 patients who relapsed and 19 patients who had CR after chemotherapy were collected for flow cytometry analysis. (B-F) The clonotype distribution of CD8⁺ T cells was associated with PD-1 expression. CD8⁺ T cells were sorted from one patient who had CR (B and C) and one relapsed patient (D and E) based on the expression of PD-1. mRNA extraction and deep sequencing of TCRβ CDR3 were performed. (B and D) Analysis of TCRβ repertoire clonal frequency in CD8⁺ (as shown above), CD8⁺ PD-1⁺ (4 × 10⁴ cells), and CD8⁺ PD-1^- (4 × 10⁴ cells) T cells. The abundances of the top 1 to 10, 11 to 20, and 21 to 50 clonotypes, as well as the remaining clonotypes, are shown. (C and E) The distribution of the top 50 most frequent clonotypes among CD8⁺ T cells was compared between the CD8⁺ PD-1⁺ populations and CD8⁺ PD-1^- populations. Each dot represents a clonotype from a newly diagnosed sample; each rhombus represents a clonotype from a sample post chemotherapy. Red represents the frequency of clonotypes in CD8⁺ PD-1⁺ > CD8⁺ PD-1^-; green represents the frequency of clonotypes in CD8⁺ PD-1^- > CD8⁺ PD-1⁺. The total frequency (Σ freq.) of the clonotypes in each population is cumulative. (F) The top 50 clonotypes in CD8⁺ T cells were frequently distributed in the CD8⁺ PD-1⁺ and CD8⁺ PD-1^- populations. Red dots represent samples collected at new diagnosis from patients who relapsed post chemotherapy; green dots represent samples collected after treatment from patients who relapsed post chemotherapy; blue triangles represent samples collected at new diagnosis from patients with sustained CR post chemotherapy; yellow triangles represent samples collected after treatment from patients with sustained CR post chemotherapy; CD8⁺ PD-1^- and CD8⁺ PD-1⁺ T cell from 5 patients who relapsed and 17 patients who had CR after chemotherapy were collected for TCRβ CDR3 sequencing. (G) Shannon index comparison between CD8⁺ PD-1⁺ and CD8⁺ PD-1^- populations. The Wilcoxon signed-rank test was used for matched paired comparisons. **P < 0.05; ***P < 0.01.