Fig. 5: TCRβ sequencing analysis reveals clonal differences following administration of anti-OX40.

TCRβ sequencing was performed on blood and TIL samples obtained before and after anti-OX40 administration. The gating strategy for the isolation of the blood and TIL subsets is outlined in Supplementary Fig. 6a, b. a Schematic of all T-cell subsets that were isolated from blood and tumor by cell sorting, followed by DNA isolation and TCRβ sequencing. b Summary of the clonality of CD4+ and CD8+ T cells in blood before anti-OX40 as well as blood and drLN at DOS in four patients. c Summary of the clonality of CD4+ TIL and CD8+ TIL subsets (DN, SP, and DP) before (pre) and after (post) anti-OX40. N = 2 pre, N = 4 post. Error bars indicate mean ± SEM in the post patients. d TCRβ repertoire overlap was calculated using the Morisita–Horn index. Overlap analysis is shown for DN CD8+ TIL compared with SP, DP, and drLN cells. The same analysis was performed for SP CD8+ TIL (with DN, DP, and drLN) and DP CD8+ TIL (with DN, SP, and drLN). Colors and symbols in (b) and (d) depict the four patients. e The top 30 clones in the DOS specimen (DN, SP, and DP) were separately compared to the same subsets pretreatment and to memory CD8+ T cells in blood at D1 and D12. Black open circles represent the blood before and after anti-OX40, orange filled circles represent the biopsy (pre) specimen, and red filled circles the DOS (post) specimen. Connecting lines indicate the presence of the same TCRβ sequence in each subset. f Red filled circles represent the number of clones among the top 30 clones in DP CD8+ TIL post treatment, that were present in the sample pretreatment. In b–f, N = 4 patients (HNOX04, HNOX05, HNOX11 and HNOX18) were analyzed. M memory, LN lymph node, DOS day of surgery.