Extended Data Figure 1. Analyses of peripheral TCR repertoire diversity at baseline and following PD-1 blockade.

a) Number of prior therapies in trial patients who were treated with nivolumab ≥ 1 yr after ASCT by best overall response to PD-1 blockade (CR n=14, PR n=18, PD n=12). b) Percentages of CD3+ and CD3− viable cells at baseline in trial patients with relapsed/refractory cHL. Viable singlet cells identified by manual gating of CyTOF data were divided according to CD3 expression (CD3−, grey and CD3+, orange, n=38). Individual samples from patients with available CyTOF files who had relapsed/refractory cHL with ≥ 1 year between nivolumab and prior myeloablative ASCT are shown (n=38) (CR n=13, PR n=15, PD n=10). c) Comparison of baseline CD3+ populations in trial patients with relapsed/refractory cHL (from b) according to their subsequent response to PD1 blockade d) Total number of TCR-seq detected clones at baseline in trial patients (from a) according to their subsequent response to PD1 blockade. e) Percentages of CD4+ (blue) and CD8+ (purple) cells at baseline in trial patients with relapsed/refractory cHL. CD3+ cells identified (from b) and divided according to CD4+ or CD8+ expression by manual gating of CyTOF data. Additional cryopreserved samples from indicated cases (*) were available for CD4+ and CD8+ sorting (n=18, 2 excluded from this analysis as no CyTOF files available). f) Comparison of baseline CD4+ populations in all trial patients with relapsed/ refractory cHL (from e) according to their subsequent response to PD1 blockade (CR, PR, PD). g) Comparison of baseline CD4+ populations in trial patients with relapsed/refractory cHL (from e*) with additional PBMC samples sorted for CD4+ and CD8+ T cells (n=18). h) Total numbers of CD4+ and CD8+ TCR-seq detected clones at baseline in trial patients (from g) according to their subsequent response to PD-1 blockade. Differences between groups in panels a, c, d, f, g and h were assessed with a Wilcoxon rank sum test of the median with two-tailed p values. i) Changes in TCR diversity from C1D1 to C4D1 in the subset of trial patients with known HRS cell expression of MHC class I and MHC class II and CD4+ and CD8+ TCRseq data (n=9). Definitions of positive (positive or decreased) and negative expression of MHC class I and class II on HRS cells previously described in (Roemer et al 2018⁴). j) Changes in TCR diversity from C1D1 to C4D1 separated by HRS cell expression of MHC class II only, samples from h. Differences in panels i and j were assessed by Wilcoxon rank sum test with one-sided p-values. k) The ratio of maximum expansion of singleton clones (0 or 1 copy at baseline)/ non-singleton clones which have 2 or more copies at baseline in patients with BOR of CR (n=9), PR (n=17) or PD (n=8) to PD-1 blockade. Only patients with all 3 timepoints are included in the analysis. Differences between groups were assessed with a Wilcoxon rank sum test of the median, two-tailed p values. (l and m) The ratio of expanded singleton / non-singleton clones from CD4+ only T cells (l) or CD8+ only T cells (m) from patients with CR, PR or PD to PD-1 blockade (n=20). Differences in panels l and m were assessed by Wilcoxon rank sum test with one-sided p-values. Graphpad Prism (v8) or R (ggplot function) was used to generate box plots (GraphPad Prism panels b, c, e-g and R panels a, d, h-m). The box corresponds to the first and third quartiles and whiskers define minimum and maximum values. Outliers beyond 1.5x IQR in R- generated plots are plotted individually.