Fig. 6. Clonal dynamics of CD8+ TCR-clonotypes before and after PD1-1 blockade.

(A) UMAP of CD8+ TILs based on intra-patient TCR clone frequency (defined through scTCR-seq) to represent clonally expanded CD8+ T cells. TCR clonotypes in 3 Rs (top) and 3 NRs (bottom) with matched biopsies collected before (pre) and (post) anti-PD1 therapy are shown. (B) Cluster distribution of the top 50 CD8+ TCR clonotypes classified as T_NExM or T_Ex, sequenced in pre-post therapy tumor biopsies from 3 Rs (top) and 3 NRs (bottom). Colors denote cell states inferred from scRNA-seq (see Fig. 2B), as delineated within the legend (bottom). (C) Overall frequencies of CD8+ TCR clonotypes, classified as Lost (orange), New (green) or Persisting (black) based on detection in pre and/or post therapy biopsies. Dots depict mean values with standard deviations, as assessed in 3 Rs (left) and 3 NRs (right) before (pre) or after (post) anti-PD-1 immunotherapy. P value reports significant differences, as established using two-tailed t-test. For each timepoint, bottom pies report the total number of TCR clonotypes for each category (Data File S8). (D) Clone size of TCR clonotypes, as classified in panel C and analyzed across 3 Rs (left) and 3 NRs (right) HNSCCs before or after neoadjuvant PD-1 blockade. Dots represent counts of individual TCR clonotypes, while boxes report 25^th-75^th percentiles with medians (horizontal bars). Whiskers: min-max values; P values indicate significant comparisons calculated with two-tailed paired t-test (for persisting clones, pre vs post) or two-tailed unpaired Welch’s t-test. (E), Bar plots depicting overall intratumoral frequencies of CD8+ TCR-clonotypes with different primary phenotypes (T_NExM: blue, T_Ex: red) among total CD8+ TILs. TCR clonotypes are classified based on their detection in pre and/or post therapy biopsies as Lost (pre-specific), New (post-specific) or Persisting (detected in both pre-post samples). Values are shown for each 3 Rs and 3 NRs with pre-post therapy assessments. Based on their size, TCR clonotypes are further divided in singletons (lightest colors), doubletons (intermediated colors) or expanded (composed by >2 cells; darkest colors Data File S9). P values indicates significant comparisons for expanded TCR clonotypes, as calculated with two-tailed unpaired Welch’s t-test (Rs vs NRs) or with two-tailed paired t-test (pre vs post). (F) Dynamics of Persisting or CD8+ TCR clonotypes with T_NExM (blue), T_Ex (red) primary phenotypes. Lines depict individual clones detected before (pre) or after (post) neoadjuvant PD-1 blockade within the TME of HNSCCs from 3 Rs (left) and 3 NRs (right). Bold lines report mean values. P values indicate significant clonal expansions or contractions, as calculated with two-tailed paired t-test.