Expansion of CD4⁺ T cells at time of response to PD-1 blockade. (A) Relative abundance of T-cell clusters defined by scRNA-seq (Figure 1C) and cytometry by time-of-flight (CyTOF) (Fig. 1G) at transient partial response to nivolumab (C2D1, C4D1) and subsequent disease progression with unsuccessful re-exposure to nivolumab (relapse, C6D1, and progression). (B) Multidimensional scaling (MDS) plot of T cells based on marker expression (CyTOF). The size of the dots indicates the number of T cells per sample. Black indicates sampling before nivolumab; green, sampling at time of transient response; red, sampling at time of relapse. (C) Quantification of regulatory T cells using CyTOF and manual gating. (D) Abundance of clonally expanded T-cell clonotypes relative to all detected T-cell clonotypes before nivolumab (%Baseline) and at relapse (relapse, C6D1, and progression). T-cell clonotypes are defined by identical complementarity defining region 3β (CDR3β) sequence. Statistical testing using Fisher’s exact test (P < .05). Gray indicates unchanged abundance; red, lower abundance at relapse; black, higher abundance at relapse; ND, not detected. (E-F) Expression of CD8, CD28, and CD57 on T cells quantified using CITE-seq. Coloring indicates whether cells belong to clonotypes that were stable (gray), contracted (red), or expanded (black) in (D). (G-H) Differentially expressed genes in T cells before and after nivolumab treatment based on scRNA-seq. Expression of IFI6 on single T cells (H) mapped to UMAP representation (Figure 1C). (I) Differential gene activity scores calculated from scATAC-seq of T cells. FC, fold change.