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Clinical Trial
. 2014 Nov 27;515(7528):568-71.
doi: 10.1038/nature13954.

PD-1 blockade induces responses by inhibiting adaptive immune resistance

Paul C Tumeh  1 , Christina L Harview  2 , Jennifer H Yearley  3 , I Peter Shintaku  2 , Emma J M Taylor  2 , Lidia Robert  2 , Bartosz Chmielowski  1 , Marko Spasic  2 , Gina Henry  2 , Voicu Ciobanu  2 , Alisha N West  2 , Manuel Carmona  2 , Christine Kivork  2 , Elizabeth Seja  2 , Grace Cherry  2 , Antonio J Gutierrez  2 , Tristan R Grogan  2 , Christine Mateus  4 , Gorana Tomasic  4 , John A Glaspy  1 , Ryan O Emerson  5 , Harlan Robins  6 , Robert H Pierce  3 , David A Elashoff  1 , Caroline Robert  4 , Antoni Ribas  1
Affiliations
Clinical Trial

PD-1 blockade induces responses by inhibiting adaptive immune resistance

Paul C Tumeh et al. Nature. .

Abstract

Therapies that target the programmed death-1 (PD-1) receptor have shown unprecedented rates of durable clinical responses in patients with various cancer types. One mechanism by which cancer tissues limit the host immune response is via upregulation of PD-1 ligand (PD-L1) and its ligation to PD-1 on antigen-specific CD8(+) T cells (termed adaptive immune resistance). Here we show that pre-existing CD8(+) T cells distinctly located at the invasive tumour margin are associated with expression of the PD-1/PD-L1 immune inhibitory axis and may predict response to therapy. We analysed samples from 46 patients with metastatic melanoma obtained before and during anti-PD-1 therapy (pembrolizumab) using quantitative immunohistochemistry, quantitative multiplex immunofluorescence, and next-generation sequencing for T-cell antigen receptors (TCRs). In serially sampled tumours, patients responding to treatment showed proliferation of intratumoral CD8(+) T cells that directly correlated with radiographic reduction in tumour size. Pre-treatment samples obtained from responding patients showed higher numbers of CD8-, PD-1- and PD-L1-expressing cells at the invasive tumour margin and inside tumours, with close proximity between PD-1 and PD-L1, and a more clonal TCR repertoire. Using multivariate analysis, we established a predictive model based on CD8 expression at the invasive margin and validated the model in an independent cohort of 15 patients. Our findings indicate that tumour regression after therapeutic PD-1 blockade requires pre-existing CD8(+) T cells that are negatively regulated by PD-1/PD-L1-mediated adaptive immune resistance.

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Figures

Extended Data Figure 1
Extended Data Figure 1. CD8 cell infiltrates in tumor biopsies
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 illustrates S100+ tumour (left of red line) and S100− stroma (right of red line). Coordinates of the invasive margin and tumour parenchyma are generated from the S100 stained image (column labeled S100) and subsequently imported into the CD8 stained image (column labeled CD8). This is followed by a deconvolution imaging algorithm of the CD8 stained image where first, all nuclei (column labeled Nuclei+) are identified and quantified, irrespective of what type of cell. This is followed by identifying CD8+ membrane (column labeled CD8+) 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 +20, clinical and radiographic examinations indicated progressive disease; at a time when CD8 T-cells expression increased in density at the invasive margin.
Extended Data Figure 2
Extended Data Figure 2. Proliferation of CD8+ T cells in regressing tumours
a, Relationship of the change in CD8+ cell density and best percent 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 tumor response and another with progression. Double positive CD8 cells (red labeled Ki67 nucleus, CD8 brown labeled membrane) are not present at baseline, but are present during tumor regression in the biopsy from a patient with a tumor response. The double positive cells are not in the biopsy of a patient with progression during treatment (no Ki-67 labeling in brown CD8 cells). Magnification, X40.
Extended Data Figure 3
Extended Data Figure 3. Granzyme B and pSTAT1 expression before and during treatment in terms of clinical response
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, X40. 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).
Extended Data Figure 4
Extended Data Figure 4. Relationship between CD8 and PD-L1 expression in terms of treatment outcome
a, Scatterplots of CD8 and PD-L1 density (cells/mm2) 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 CD8highPD-L1high quadrant, in both tumour and invasive margin, were predominantly derived from the Response group. Samples present in the CD8lowPD-L1low 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, X20. 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 coexpression of CD8 and PD-1 on individual cells within the tumour microenvironment. CD8+ cells were 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 2 antigens were observed when the two channels are combined, yielding yellow signal in areas of colocalization.
Extended Data Figure 5
Extended Data Figure 5. PD-L1 expression and relationship with T cell infiltration
a, Multiplexed chromogenic staining of SOX-10 (red nucleus) and PD-L1 (brown membrane) to evaluate PD-L1 expression on melanoma cells, lymphocytes, and macrophages within the tumour microenvironment. SOX-10 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, X40. b, Principal component analysis (PCA) to decompose the variance for the markers (CD8+, PD-1+, PD-L1+, and CD4+ cell densities (cells/mm2) 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 center (***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.
Extended Data Figure 6
Extended Data Figure 6. Clonality of the T-cell repertoire and density of T-cell infiltration (TIL) in terms of clinical response
a, High throughput quantitative sequencing of the rearranged TCR beta 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 supplementary methods for further detail). The axes cross at the median value for clonality and TIL infiltration. TIL infiltration and TIL repertoire clonality were found to be independent in this cohort (R2 = 0.04). Progressors were associated with lower levels of TIL infiltration and lower TIL clonality (i.e., a more diverse TIL repertoire); all patients with below-median clonality and TIL infiltration progressed. b, The uniqueness of the CDR3 TCR sequences enable tracking of clonal expansion or contraction clones in serially sampled tumours. Representative scatterplot of clones from a responding tumour serially sampled before and during aPD-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 2x change in frequency from baseline must have also met significance using a Fisher Exact test of the clone before and during aPD-1 and the full set of clone reads, followed by Storey’s Qvalue for false discovery rate. c, Clonal expansion in terms of clinical response (Response n=6, Progression n=5) **P= 0.006
Figure 1
Figure 1. Immunohistochemical analysis of CD8+ T cells in samples obtained before and during pembrolizumab treatment
a and b, Examples of CD8 expression in melanoma tumours serially biopsied before PD-1 blocking treatment (Tx) and 20–60 days after treatment began (Days + 20–60) from a patient in the Response (a) and Progression (b) groups. Red line separates tumour parenchyma (below line) and invasive margin (above line). Magnification, X20. c and d, CD8+ cell density at the tumour center and invasive margin in samples from all Responders (c, n = 13) and Progressors (d, n = 12) who received a biopsy before and during treatment.● =complete response, ○ =partial response, △ = delayed response.
Figure 2
Figure 2. Regressing tumours during treatment are associated with proliferating CD8+ T cells that localize to the tumour
a, Representative example of CD8/Ki67 chromogenic double staining from a sample obtained during tumour regression shows double positive CD8 cells localized to the tumour parenchyma. The red line separates the invasive margin (above line) and tumour (below line). b, Top: Representative single positive quiescent CD8+ brown cells (no Ki-67 labeling) from the invasive margin. Bottom: Representative double positive cells (red labeled Ki67 nucleus, CD8 brown labeled membrane) with characteristic chromatin patterns associated with subphases of mitosis. Magnification, X40.
Figure 3
Figure 3. Baseline density, location, and proximity of CD8+, PD-1+, PD-L1+, and CD4+ cells, and T cell repertoire according to treatment outcome
a, Melanoma samples collected before treatment with PD-1 blocking therapy were assessed for CD8 (Response n=22, Progression n=24), PD-1 (Response n=19, Progression n=21), PD-L1 (Response n=17, Progression n=21), and CD4 (Response n=19, Progression n=18) density by quantitative immunohistochemistry in the tumour compartment and at the invasive margin. **P < 0.01, ***P < 0.001, ****P < 0.0001. b, Examples of the relative proximity of PD-1 and PD-L1 expressing cells in representative baseline samples from a responder and a progressor. c, Proximity analysis of PD-1 and PD-L1 based on multiplex quantitative immunofluorescence in baseline tumour samples (Response n=11, Progression n=11). **P = 0.005. d, Results of TCR sequencing performed on 25 whole tumour samples taken at baseline (Response n=12, Progression n=11). **P = 0.004. △ = Delayed Response.
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