Changes in T-cell frequencies in blood after administration of pembrolizumab. Frequencies of cell populations among PBMCs collected before pembrolizumab administration (baseline), on C3D1 or C7D1 were evaluated. (A) Frequency of PD-1⁺ cells at baseline, gated under the T-cell populations indicated on the x-axis. Color key shows patients by ID number. (B) Frequency of PD-L1⁺ cells among live cells. Frequency of T cells as analyzed by flow cytometry (C) (CD3⁺) or TCR sequencing (D) (TCRseq) (number of cells expressing TCR/number of total nucleated cells). (E) Changes in T-cell subset ratios (% CD3⁺CD4⁺/% CD3⁺CD8⁺). Statistical significance was evaluated by Wilcoxon matched-pairs signed-rank test. P < .05 was considered significant. *P = .0391.