Pembrolizumab-induced alterations in T-cell repertoire. TCR Vβ chains of peripheral blood T cells collected before administration of pembrolizumab (baseline) and on C3D1 or C7D1 were sequenced. (A) Clonality of T cells was calculated using the Simpson index (scores range from 0 to 1); a score of 1 indicates a monoclonal population. Statistical significance was evaluated by Wilcoxon matched-pairs signed-rank test. (B) Top 500 clones with the highest total productive frequencies (the sum of frequencies found in each sample) were evaluated to identify shared clones. The plot displays the number of patients (n = 9, y-axis) or samples sequenced (n = 22, x-axis), in which individual clones were found. The size of the bubbles indicates the total frequency of clones. (C) Number of clones for each patient that were significantly expanded at C3D1 and C7D1 compared to baseline. (D) Changes in the abundance of unique TCR Vβ sequences after 2 cycles of pembrolizumab treatment were analyzed (C3D1 vs baseline). Only clones with a minimum cumulative abundance of 10 were included in the analysis. Significantly expanded or contracted clones are denoted in orange and blue, respectively. The clones analyzed at C3D1 were evaluated for their presence at C7D1, where available (titled in red), and those that were also found at C7D1 were marked by a black circle. Significance was evaluated by the binomial method (two-sided), and false discovery rates were controlled by using the Benjamini-Hochberg method. Differential abundance of clones was considered significant if P ≤ .01. NA, not applicable.