A transcriptionally and functionally distinct PD-1+ CD8+ T cell pool with predictive potential in non-small-cell lung cancer treated with PD-1 blockade - PubMed (original) (raw)

. 2018 Jul;24(7):994-1004.

doi: 10.1038/s41591-018-0057-z. Epub 2018 Jun 11.

Viktor H Koelzer 3 4, Petra Herzig 5, Andreas Roller 6, Marcel Trefny 5, Sarah Dimeloe 7, Anna Kiialainen 6, Jonathan Hanhart 3, Catherine Schill 8, Christoph Hess 7, Spasenija Savic Prince 9, Mark Wiese 10, Didier Lardinois 10, Ping-Chih Ho 11, Christian Klein 12, Vaios Karanikas 12, Kirsten D Mertz 3, Ton N Schumacher 13, Alfred Zippelius 14 15

Affiliations

A transcriptionally and functionally distinct PD-1+ CD8+ T cell pool with predictive potential in non-small-cell lung cancer treated with PD-1 blockade

Daniela S Thommen et al. Nat Med. 2018 Jul.

Abstract

Evidence from mouse chronic viral infection models suggests that CD8+ T cell subsets characterized by distinct expression levels of the receptor PD-1 diverge in their state of exhaustion and potential for reinvigoration by PD-1 blockade. However, it remains unknown whether T cells in human cancer adopt a similar spectrum of exhausted states based on PD-1 expression levels. We compared transcriptional, metabolic and functional signatures of intratumoral CD8+ T lymphocyte populations with high (PD-1T), intermediate (PD-1N) and no PD-1 expression (PD-1-) from non-small-cell lung cancer patients. PD-1T T cells showed a markedly different transcriptional and metabolic profile from PD-1N and PD-1- lymphocytes, as well as an intrinsically high capacity for tumor recognition. Furthermore, while PD-1T lymphocytes were impaired in classical effector cytokine production, they produced CXCL13, which mediates immune cell recruitment to tertiary lymphoid structures. Strikingly, the presence of PD-1T cells was strongly predictive for both response and survival in a small cohort of non-small-cell lung cancer patients treated with PD-1 blockade. The characterization of a distinct state of tumor-reactive, PD-1-bright lymphocytes in human cancer, which only partially resembles that seen in chronic infection, provides potential avenues for therapeutic intervention.

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Conflict of interest statement

Competing financial interests:

A.R., A.K., C.K., V.K. are employed by Roche. A.Z. received research funding from Roche. Part of the work described in this manuscript is the subject of a patent application co-owned by NKI-AVL and the University of Basel. Based on NKI-AVL and the University of Basel policy on management of intellectual property, D.S.T., V.H.K., K.D.M., A.Z. and T.N.S. would be entitled to a portion of received royalty income.

Figures

Figure 1

Figure 1. Co-receptor expression, functionality and tumor reactivity of CD8+ PD-1+ TIL populations in NSCLC.

(a) Gating strategy of CD8+ TIL subsets according to PD-1 MFI for subgroup analysis, one exemplary NSCLC specimen is depicted. (b) Co-expression of other immune checkpoints on PD-1 subgroups in NSCLC specimens (n=24). Lines and boxes represent mean and SD, respectively. (c) Gating strategy to identify PD-1 subsets within peripheral blood CD8+ T cells of healthy donors and within intratumoral T cells in NSCLC. (d) Effector cytokine secretion of sorted PD-1 subsets with and without anti-CD3/anti-CD28 stimulation measured by bead-based immunoassay (mean and SEM of three donors). (e) Clonality of the TCR repertoire of PD-1T, PD-1N and PD-1- TILs. Data from 11 NSCLC specimens are shown as box-and whisker-plots (the lines indicate median values, the boxes interquartile range (IQR) values and the whiskers 1.5IQR values as calculated by Tukey) (left). ***P < 0.001 by one-way analysis of variance (ANOVA). The average relative abundance of the most frequent TCRβ clonotype, the second most frequent, the 3rd to 30th most frequent, and the remaining clonotypes are shown for all 11 donors (right). (f) Frequency of the 30 most abundant TCR sequences of the PD-1T subset within PD-1T, PD-1N and PD-1- populations. Shown are the mean percentage and standard deviation of all 11 donors. ***P < 0.001, ****P < 0.0001 by one-way ANOVA. (g) Expression of CD137 in PD-1 subgroups (n=24). Lines and boxes represent mean and SD, respectively. (h) Flow cytometry plot and quantification of pre-/postexpansion PD-1 expression and IFN-γ secretion of TILs expanded from the three sorted PD-1 subsets (n=3 NSCLC specimens). PD-1 MFI was analyzed within CD3+CD8+ cells from each expanded subset. Bar graphs indicate mean and SEM. ***P < 0.001 by two-way ANOVA. (i) IFN-γ expression of expanded PD-1T, PD-1N and PD-1- TILs from the eight donors upon co-culture with autologous digests. The difference with and without HLA class I blocking antibody is depicted for each individual donor (left) and T cell only and PMA/ionomycin controls are shown for all samples (mean and SD, right).

Figure 2

Figure 2. Gene expression profile of sorted PD-1T, PD-1N and PD-1- TILs from NSCLC specimens.

(a) Principal component analysis (left) and bar plots for the means of the PC1 and PC2 values (right) for sorted PD-1T, PD-1N, PD-1- TILs (n=11) and healthy donor effector memory (EM) T cells (n=4). Error bars represent the SEM. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. (b) Significantly regulated genes between the three PD-1 subsets. Significance was determined as Benjamini-Hochberg FDR<0.01 and log2 fold change ≥1. (c) Clustering analysis for genes distinguishing PD-1T from PD-1N and PD-1- subsets. (d) Selected clusters showing up- or downregulation of key biological processes (Gene ontology (GO) terms). (e) Biological processes (GO terms) enriched in clusters 5 and 7. Numbers in parentheses indicate the number of genes within each GO term. (f) Increased expression of the proliferation marker Ki67 (MKI67) at the mRNA level (left, n=11), and at the protein level as assessed by intracellular staining of ex vivo tumor digests (n=5, middle) and sorted PD-1 subsets that were cultured for 48 hrs (n=3, right). The lines in the box-and whisker-plot indicate median values, the boxes IQR values and the whiskers 1.5IQR values as calculated by Tukey. The dot plots and bar graphs represent mean and SEM. *P < 0.05, ***P < 0.001, ****P < 0.0001 in (b), (c) and (d) by one-way ANOVA.

Figure 3

Figure 3. PD-1T TILs show overexpression of inhibitory receptors, but display a key gene signature distinct from exhausted T cells in murine chronic infection and cancer.

(a) Volcano plot of up- or downregulated genes between PD-1T and PD-1N TILs, with a set of genes encoding inhibitory receptors and molecules involved in proliferation and differentiation annotated (n=11 tumor specimens). Significance was determined as Benjamini-Hochberg FDR<0.01 and log2 fold change ≥1. (b) Surface expression of indicated receptors on PD-1T, PD-1N, and PD-1- gated TILs in tumor digests (n=8), as determined by flow cytometry. (c) Expression of the ‘exhaustion genes’ described in Zheng et al. in the indicated PD-1 subsets (n=11). The lines in the box-and whisker-plots indicate median values, the boxes IQR values and the whiskers 1.5IQR values as calculated by Tukey. (d) Surface expression of CD27 and KLRG1 on PD-1T, PD-1N, and PD-1- gated TILs in tumor digests (n=8), as determined by flow cytometry. Each dot represents one patient. Bar graphs indicate mean and SEM. **P < 0.01, ***P < 0.001, ****P < 0.0001 in (b), (c) and (d) by one-way ANOVA. (e) Venn diagram comparing the overlap of the differentially expressed genes between PD-1T and PD-1N TILs with the genes derived from the chronic LCMV and the early/late-stage tumor signatures, respectively. (f+g) Gene set enrichment analysis (GSEA) of published data sets from exhausted T cells in (f) chronic murine LCMV infection (Crawford et al.) and (g) murine tumors (Schietinger et al.) within the gene signatures derived from the differently expressed genes between PD-1T and EM T cells or PD-1N and EM T cells (n=11 tumor specimens and 4 healthy donors). Statistical significance was determined by permutation testing with NES (normalized enrichment score).

Figure 4

Figure 4. Alterations in glucose, lipid and mitochondrial metabolism in PD-1T TILs.

(a) Representative histograms of metabolic parameters analyzed by flow cytometry in T cells of one donor (from n=3 independent experiments). (b) Glucose uptake (2-NBDG), (c) lipid content (Bodipy 493), uptake (Bodipy 500) and CD36 expression, and (d) mitochondrial mass (indicated by MTG), membrane potential ΔΨm (MTR), and normalized ΔΨm (MTR/MTG ratio) of PD-1T, PD-1N and PD-1- gated TILs in 11 tumor digests. Shown are mean and SD. *P < 0.05, ****P < 0.0001 in (b), (c) and (d) by one-way ANOVA. (e) Representative electron micrographs of sorted PD-1T and PD-1N T cells and their randomly selected mitochondrial ultrastructures. Analysis of PD-1- T cells or of independent biological replicates was precluded by limited cell numbers. Scale bars, 500 nm. Quantification of mitochondria numbers per cell, numbers of cristae in mitochondria and total cristae length normalized to the surface area of mitochondria using Image J. Sample identity was blinded to the analyser and at least 10 randomly selected TILs in each group were quantified (PD-1T: 10, 27, and 21 TILs; PD-1N: 15, 23, and 12 TILs for each separate analysis). Data represent mean ± SD. *P < 0.05, ***P < 0.001 by Mann-Whitney test.

Figure 5

Figure 5. PD-1T TILs display a fixed state of dysfunction.

(a) Expression of Tim-3 and Lag-3 in IL-2 expanded sorted TILs (n=3). Shown are mean and SEM. (b) IL-10 expression by indicated cell subsets in tumor digests and correlation of IL-10 expressing cells with the number of PD-1T TILs (n=8). Lines in box-and-whisker-plots indicate median values, boxes indicate IQR values and whiskers minimum and maximum values. R2 = 0.61 was calculated using linear regression analysis. (c) PD-1 upregulation in sorted TIL subsets after withdrawal of IL-2 and short-time IL-10 exposure (n=3). Bar graphs indicate mean and SEM. *P < 0.05, **P < 0.01 by one-way ANOVA. (d) IFN-γ production of IL-10 expanded PD-1T, PD-1N, PD-1- sorted TILs (n=3). Bar graphs indicate mean and SEM. (e) IL10R and perforin expression in IL-2 expanded TIL subsets with and without IL-10 stimulation.

Figure 6

Figure 6. CXCL13 expression of PD-1T TILs and predictive potential for response to PD-1 blockade.

(a) Expression of indicated inflammatory cytokines and chemokines in sorted PD-1T TILs after 24 hrs of resting by bead-based immunoarray. Shown are mean and SEM of 3 donors. (b) Migration of peripheral blood CXCR5+ CD8+, CD4+ and CD19+ and CXCR5- CD3+ immune cell subsets in response to CXCL13. Shown are the mean and SEM from three donors. (c) Digital markup images showing the color deconvolution of immunohistochemistry staining of CD8 (red) /PD-1 (brown) double positive cells. (d) Immunohistochemical analysis of tertiary lymphoid structures (TLS) in lung cancer. Distribution of TLS in the tumor (large image, arrows indicate TLS, H&E). TLS show accumulation of CD8/PD-1T double positive and CD4+ T cells in the periphery of the follicular structure. Interspersed Bcl6+ cells likely represent follicular helper T cells. A well-formed CD21+ follicular dendritic cell network with strong accumulation of B cells is found in the center of the follicle. Digital markup image showing the color coding of CD8+ (red), PD-1+ (green), CD8+PD-1+ (yellow) and double negative (blue) cells in the TLS. (e) Digital markup images showing the localization of CD8+, PD-1+ and CD8+PD-1+ cells in five different NSCLC specimens. (f) Quantification of PD-1T TILs localized within and outside of TLS in NSCLC specimens by digital image analysis (n=10). Shown are mean and SD. **P < 0.01 by Mann-Whitney test. (g) Percentage of PD-1T TILs per total cells or number of PD-1T cells per mm2 in responders (n=7) and non-responders (n=14) to PD-1 blockade. Shown are mean and SD. ****P < 0.0001 by Mann-Whitney test. (h) Overall survival of patients with tumors harboring more or less than 1% of PD-1T cells (n=21). P value was determined by log-rank test. (i) Duration of response in the seven responding patients.

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