A) Overview of cohort. Blood samples were collected
pre-treatment (baseline, 2–29 days before surgery), at time of surgery,
and at follow-up (27–38 days post-surgery) from patients with HNSCC that
received anti-PD-L1 treatment prior to surgery (n = 10). Additionally, tumor
samples were collected at time of surgery. For a few patients, additional
samples were collected (see complete overview in Figure S6A). Samples were analyzed
by mass cytometry. B) Heatmap of markers used for CD8+ T cell
clustering. Scaled median expression per marker is shown for cluster annotation.
C) UMAP of non-naïve CD8+ T cell clusters and expression
for a subset of markers. D) Frequencies of non-naïve CD8+ T
cell clusters. E) Paired differential abundance analysis of
non-naïve CD8+ T cell subsets between blood at time of surgery and
baseline (n = 10) (generalized linear mixed models). The log2 fold changes are
plotted against the negative log10(nominal p-values). Colors indicate if cell
populations are significantly more abundant in blood at time of surgery (purple)
or baseline (blue) or not differentially abundant (False, grey) after
Benjamini-Hochberg correction, FDR < 0.1. F) Cluster 5 and 9
abundances (as percentage of non-naïve CD8+ T cells) in paired blood
samples at time of surgery and baseline. P-values obtained by generalized linear
mixed models. G) Paired differential abundance analysis of
non-naïve CD8+ T cell subsets between blood at time of follow-up and at
baseline (n = 7) (generalized linear mixed models). See color scheme for Figure 6C. H) Cluster 2, 5, and
9 abundances (as percentage of non-naïve CD8+ T cells) in paired blood
samples at follow-up and baseline. P-values obtained by generalized linear mixed
models. I-J) Percentage of cluster 2 and 5 proliferating cells in
(I) paired blood samples at time of surgery and baseline and
(J) in blood and tumor at time of surgery. P-values obtained by
paired Wilcoxon Rank Sum Test. K) Spearman correlation between
cluster 16 and 18 in LN (MIBI data) and cluster 5 and 9 in blood (CyTOF data) at
time of surgery. See also Figure S5.