Fig. 5. Quality and quantity of TILs in pre-treatment HNSCC associate with pathologic tumor response after neoadjuvant PD-1 blockade.

(A) Summary of quantitative and qualitative differences in putative tumor-reactive CD8+ T_Ex-TILs detected before or after neoadjuvant PD-1 blockade, as established in patients with available paired scRNA-seq (see Fig. 2A). Box plots - ratios (Data File S4) calculated using the frequencies of putative-tumor reactive CD8+ T_Ex and memory CD8+ T_NExM (left) or expressing the relative proportion of cytotoxic terminal exhausted CD8+ TILs (T_TE-CTX) over other CD8+ T_Ex subsets (right). Dots - individual values in Rs (n=3, circles) and NRs (n=3, diamonds), with lines connecting pre-post matched evaluations for each patient. P values: significant comparisons calculated with two-tailed unpaired t-test (R vs NR) or with two-tailed paired ratio t-test (pre vs post). (B) Enumeration of tissue-resident (CD103+ PD-1+) CD3+ CD8+ T cells infiltrating HNSCC specimens collected at pre-treatment timepoint from 35 patients, classified based on the extent of pathological tumor response (pTR) assessed after one (lighter shaded) or two (darker shaded) doses of neoadjuvant anti-PD-1 (cohort 1 and 2, Data File S6). Significance was calculated between Rs and NRs (pTR-1/2 vs pTR-0) or between pTR2 and pTR-0 with two-tailed Mann-Whitney test. (C) The same analysis was performed for 17 patients with available tumor specimens collected before or after two doses of pembrolizumab (cohort 2, Data File S6). Quantification of TILs was performed through immunofluorescent staining, analyzed as reported in Materials and Methods section. P values: significant comparisons calculated with two-tailed Mann-Whitney test (for group comparisons) or with two-tailed paired t-test (for pre vs post). (D) Analysis of gene-expression profile of HNSCC biopsies collected before or after neoadjuvant PD-1 blockade in patients treated with a single dose of pembrolizumab (cohort 1), as previously reported (3). Tumor bulk RNA-seq was analyzed as described in Materials and Methods section, to quantify CD8+ T cells (top) or to evaluate the level of expression of ZNF683 and cytotoxicity (CTX) genes before (pre, left) or after (post, right) neoadjuvant immunotherapy (Data File S7). The same analysis is performed on RNA-seq data available from 11 HNSCC biopsies collected before or after 3 doses of nivolumab (E), as reported in (30). P values: significant comparisons calculated with two-tailed Mann-Whitney test. In all the panels, dots represent values from individual patients, with colors depicting the level of pTR (panels A-D) or radiographic reduction of tumor (panel E, as established in (30)) measured after neoadjuvant immunotherapy. For column plots: boxes: 25^th-75^th percentiles; whiskers: min-max values; horizontal lines: media values.