PD-1 blockade induces responses by inhibiting adaptive immune resistance

a, Segmentation of the invasive margin and tumour parenchyma using S100 and CD8 chromogenic staining. Low magnification (top row) and high magnification (bottom row) are shown. The red dotted line demarcates S100⁺ tumour (left of red line) from S100⁻ stroma (right of red line). Coordinates of the invasive margin and tumour parenchyma are generated from the S100 stained image (column labelled ‘S100’) and subsequently imported into the CD8 stained image (column labelled ‘CD8’). This is followed by a deconvolution imaging algorithm of the CD8 stained image where first all nuclei (column labelled ‘nuclei+ cells’) are identified and quantified, irrespective of what type of cell. This is followed by identifying CD8⁺ membrane (column labelled ‘CD8⁺ cells’) for cell quantification and analysis. b, CD8⁺ T-cell kinetics within the tumour microenvironment in a serially sampled tumour responding to PD-1 blocking therapy. Example of radiographic, clinical, and CD8 IHC in a serially sampled melanoma tumour of the left chest wall that was obtained from a patient with a delayed response. On day +27, clinical and radiographic examinations indicated progressive disease; at a time when CD8⁺ T cells expression increased in density at the invasive margin.

a, Relationship of the change in CD8⁺-cell density and best percentage change in tumour size in serially sampled tumours that were assessed using quantitative immunohistochemistry and CT scan measurements (n = 18, Spearman r = −0.75, P = 0.0002). b, CD8⁺-cell density and Ki67⁺/CD8⁺-cell density in the response group before treatment (n = 11, empty circles) and during treatment (n = 17, filled circles) and the progression group before treatment (n = 9, empty triangles), and during treatment (n = 15, filled triangles). c. Representative examples of CD8/Ki67 chromogenic double staining from a biopsy of a patient with a tumour response and another with progression. Double-positive CD8 cells (red, labelled Ki67 nucleus; brown, CD8 labelled membrane) are not present at baseline, but are present during tumour regression in the biopsy from a patient with a tumour response. The double-positive cells are not in the biopsy of a patient with progression during treatment (no Ki67 labelling in brown CD8 cells). Magnification, ×40.

a, Representative examples of granzyme B expression according to clinical response. b, Samples collected during PD-1 blocking therapy were evaluated for granzyme B signal (response n = 13, progression n = 12) using quantitative immunohistochemistry. ****P < 0.0001. c, Representative example of the proximity between CD8⁺, PD-1⁺, PD-L1⁺ and pSTAT1⁺ cells in the tumour microenvironment in a sample obtained before treatment from the response group. Magnification, ×40. 2 µM serial cut tissue sections were stained for CD8, PD-1, PD-L1 and pSTAT1. d, Localization of CD8⁺ and pSTAT1⁺ cells in samples obtained before treatment from a biopsy in a patient with response and two patients with progression on anti-PD-1 therapy (± a CD8 presence). The biopsy from a patient with disease progression had a moderate presence of CD8 cells that did not show pSTAT1 expression in the area. e, Using quantitative IHC analysis, the response group was associated with significantly higher expression of pSTAT1⁺ at the invasive margin before and during treatment (response n = 16, progression n = 18, P = 0.002 for pre-treatment biopsies; and response n = 13, progression n = 12, P < 0.0001 for post treatment biopsies). Within the response group, pSTAT1 expression was significantly higher during treatment when compared to baseline (P = 0.022).

a, Scatterplots of CD8 and PD-L1 density (cells mm⁻²) using a log(x+1) scale are shown in samples obtained at baseline stratified by treatment outcome. Reference ‘cut-points’ for CD8 and PD-L1 densities were based on the median value for each marker across the entire cohort. Samples present in the CD8^highPD-L1^high quadrant, in both tumour and invasive margin, were predominantly derived from the response group. Samples present in the CD8^lowPD-L1^low quadrants were significantly associated with the progression group (response n = 17, progression n = 21, P < 0.001 at both the invasive margin and tumour). b, CD8, PD-1 and PD-L1 expression before treatment in terms of clinical response. Representative examples of the proximity between CD8⁺, PD-1⁺, PD-L1⁺ cells in the tumour microenvironment in pre-treatment samples obtained from a patient with a tumour response and progression. Magnification, ×20. 2 µM serial cut tissue sections were stained for CD8, PD-1 and PD-L1. c, Multiplexed immunofluorescence staining of CD8 and PD-1 to evaluate the relative co-expression of CD8 and PD-1 on individual cells within the tumour microenvironment. CD8⁺ cells were detected using AlexaFluor 488 staining in the green channel. PD-1+ cells were detected suing AlexaFluor 594 staining in the red channel. High levels of co-expression of the two antigens were observed when the two channels are combined, yielding yellow signal in areas of co-localization.

a, Multiplexed chromogenic staining of SOX10 (red nucleus) and PD-L1 (brown membrane) to evaluate PD-L1 expression on melanoma cells, lymphocytes and macrophages within the tumour microenvironment. SOX10 is a transcription factor that is melanoma-cell specific. Representative high power fields of double-positive cells (yellow arrows) show melanoma cells expressing PD-L1 and single-positive PD-L1 cells comprising of lymphocytes (high nuclear:cytoplasmic ratio, red arrows) and macrophages (low nuclear:cytoplasmic ratio, green arrows) in three responders from samples obtained during tumour regression. Magnification, ×40. b, Principal component analysis to decompose the variance for the markers (CD8⁺-, PD-1⁺-, PD-L1⁺- and CD4⁺-cell densities (cells mm⁻²)) separately in the tumour and in the invasive margin. The first principal component accounted for the majority of the variability in the four markers—69.6% and 57.1% in the tumour centre (***P = 0.001) and the invasive margin (****P < 0.0001), respectively. Principal component scores for the first principal component were compared between response groups with Wilcoxon rank-sum tests.

a, High-throughput quantitative sequencing of the rearranged TCR β genes using the ImmunoSeq assay (response n = 13, progression n = 12). ***P = 0.005 by Fisher’s exact test. The x-axis represents clonality of the T-cell repertoire (1 − Pielou’s evenness) and the y-axis represents the density of tumour-infiltrating T cells (estimated TCR gene rearrangements per diploid genomes, see Methods for further details). The axes cross at the median value for clonality and T-cell infiltration. T-cell infiltration and T-cell infilitration repertoire clonality were found to be independent in this cohort (R² = 0.04). Progressors were associated with lower levels of T-cell infiltration and lower T-cell infiltration clonality (that is, a more diverse T-cell infiltration repertoire); all patients with below-median clonality and T-cell infiltration progressed. b, The uniqueness of the CDR3 TCR sequences enable tracking of clonal expansion or contraction in serially sampled tumours. Representative scatterplot of clones from a responding tumour serially sampled before and during anti-PD-1 treatment. (1 dot = 1 unique clone: green, expanded; grey, stable; red, contracted). The x- and y-axes represent the relative abundance of each clone before and during treatment, respectively. Clones that met a 2× change in frequency from baseline must have also met significance using a Fisher exact test of the clone before and during anti-PD-1 treatment and the full set of clone reads, followed by Storey’s Q value for false discovery rate. c, Clonal expansion in terms of clinical response (response n = 6, progression n = 5). **P = 0.006.