Nature Immunology: doi: /ni Supplementary Figure 1. RNA-Seq analysis of CD8 + TILs and N-TILs.
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1 Supplementary Figure 1 RNA-Seq analysis of CD8 + TILs and N-TILs. (a) Schematic representation of the tumor and cell types used for the study. HNSCC, head and neck squamous cell cancer; NSCLC, non-small cell lung cancer; TIL, tumor-infiltrating lymphocyte; N-TIL, non-tumor-infiltrating lymphocyte. (b) Minus-average (MA) plots illustrate differentially expressed genes (red dots) for pairwise comparison of lung CD8 + N-TILs versus NSCLC CD8 + TILs (left), comparison of CD8 + TILs from NSCLC adenocarcinoma versus NSCLC squamous carcinoma (center), comparison of CD8 + TILs from HNSCC HPV-positive versus HPV-negative tumors (right) (DESeq2 analysis, Benjamini-Hochberg adjusted P <.5) (Online Methods and Supplementary Table 3). (c) Uupervised hierarchical clustering of the tracriptomes of all CD8 + N-TILs (grey) and CD8 + TILs from NSCLC adenocarcinoma (red) and squamous carcinoma (pink), HPV-negative (light blue) and HPV-positive (dark blue) HNSCC, based on the expression of genes with the highest variance (n = 1); each line represents an independent sample. (d) t-sne plots of CD8 + T cell core tracriptome (symbol) from N-TILs and TILs of indicated cancer subtypes. Color scheme is identical to (c). Data are from one experiment (b-d). Nature Immunology: doi:1.138/ni.3775
2 Supplementary Figure 2 Heterogeneity in the expression of molecules that are targets of immunotherapy. RNA-Seq analysis (row-wise normalized counts; bottom key) of various tracripts (left margin; one per row) in CD8 + TIL from patients with HNSCC (one per column); above, CD8 + TIL deity (top row; top left key) and tumor stage (bottom row; far right key) for each patient. Data are from one experiment. Nature Immunology: doi:1.138/ni.3775
3 Expression (normalized) Expression (normalized) PDCD1 expression (normalized) PD-1 a b 1.2 PBMC Lung N-TIL. 7.3 NSCLC TIL 2.5 r =.8182 P = Number of PD1 + cells 53.4 CD TIL lo TIL int TIL hi c PDCD1 4 r = Age (years) Male Female Adeno Sq I II III Tumor stage I Performance status Current Ex Never Smoking status TNFRSF9 4 r = Age (years) Male Female Adeno Sq I II III I Current Ex Never Tumor stage Performance status Smoking status Supplementary Figure 3 Correlation of the abundance of PDCD1 and 4-1BB tracripts with clinical and pathological characteristics of patients with NSCLC. (a) Correlation of PDCD1 tracript expression (log 2 normalized counts) in NSCLC CD8 + TIL and the average number of tumorinfiltrating PD-1 + cells (quantified by immunohistochemistry). Each symbol represents an individual patient (n = 1) (r value and P value as in Fig. 3c,d). (b) Flow-cytometry analysis of the expression of PD-1 versus that of CD8 in live and singlet-gated CD45 + CD3 + T cells obtained from matched PBMC, lung N-TILs and NSCLC TILs (above plots) from the same patient. (c) Correlation of age of NSCLC patients with PDCD1 or 4-1BB tracript expression (log 2 normalized counts) in CD8 + TILs (left) (r value as in Fig. 3c,d). Bar graphs show PDCD1 or 4-1BB tracript expression in CD8 + TILs from patient groups categorized based on gender (first), tumor histology (second), tumor stage (third), performance status (fourth) and smoking status (fifth) of NSCLC patients; adenocarcinoma (Adeno), squamous carcinoma (Sq). Each symbol represents an individual patient (a,c); small horizontal lines are the mean (± s.e.m.). P value iignificant (Kolmogorov-Smirnov test). Data are from ten experiments (a) or one experiment (c) or representative of six experiments (b). Nature Immunology: doi:1.138/ni.3775
4 Supplementary Figure 4 TIL status of patients with NSCLC. Graph shows the average number of CD8α + cells per high power field (HPF) in tumor samples from each NSCLC patient (Online Methods). Nature Immunology: doi:1.138/ni.3775
5 CD9 CD49a KLRG1 CD2L CCR7 Percentage -Log(P value) a CAST CX3CR1 c TNF Reduced cell movement S1A TIMP1 S1PR1 Upregulated Cytokine G-protein coupled receptor Peptidase Downregulated -Log(P value) Downregulated Other b * % of CD * % of CD49a * % of KLRG * % of CD2L % of CCR CD8 + CD13 - TIL 17.4 CD CD8 + CD13 + TIL Supplementary Figure 5 Pathways for which CD8 + TILs from NSCLC TIL hi tumors show enrichment, and phenotype of CD8 + CD13 + TILs. (a) Ingenuity pathway analysis of genes downregulated in CD8 + TILs from NSCLC TIL hi tumors relative to their expression in TIL lo tumors (blue), encoding molecules associated with tissue egress (shape indicates function (key)). (b) Flow-cytometry analysis of the expression of CD9, CD49a, KLRG1, CD2L or CCR7 versus that of CD13 in live and singlet-gated CD45 + CD3 + CD8 + T cells obtained from NSCLC TILs (left); frequency of CD13 + CD8 + or CD13 CD8 + TILs (n = ) that express the indicated surface marker (right). * P =.25 (CD9), P =.25 (CD49a), P =.1 (KLRG1), P =.21 (CD2L) (paired Student s two-tailed t-test). (c) Analysis of canonical pathways from the Ingenuity pathway analysis database (horizontal axis; bars in plot) for which CD8 + TILs from NSCLC TIL hi Nature Immunology: doi:1.138/ni.3775
6 tumors show enrichment (presented as in Fig. 2a) relative to their expression in TIL lo tumors (P values as in Fig. 2a). Each symbol (b) represents an individual sample; small horizontal lines indicate the mean (± s.e.m.). Data are from one experiment (a,c) or from six experiments (b). Nature Immunology: doi:1.138/ni.3775
7 Supplementary Figure CD13 (ITGAE) status of CD8 + TILs. (a) Correlation of ITGAE tracript expression (log 2 normalized counts) in NSCLC CD8 + TIL and the average number of tumorinfiltrating CD13 + cells (quantified by immunohistochemistry). Each symbol represents an individual patient (n = 1) (r value and P value as in Fig. 3c,d). (b) Classification of tumors on the basis of the expression of ITGAE (CD13) tracripts (normalized counts) in CD8 + TILs from each NSCLC patient (Online Methods). Data are from ten experiments (a) or one experiment (b). Nature Immunology: doi:1.138/ni.3775
8 Supplementary Figure 7 Deity of CD13 + cells is predictive of survival in lung cancer. a) Correlation of GZMB tracript expression (log 2 normalized counts) in NSCLC CD8 + TIL and the average number of tumor-infiltrating GZMB + cells (quantified by immunohistochemistry, n = 1) (r value and P value as in Fig. 3c,d). (b) Immunohistochemistry microscopy of cytokeratin, CD13, CD8α, PD-1 and GZMB (above images) in CD13 lo and CD13 hi NSCLC tumors (left margin). Scale bars, 1 μm. (c) Frequency of CD8 + TILs expressing the indicated molecules that are either CD13 + or CD13 - (n = 9-19). * P =.3 for GZMA and P =.29 for GZMB (paired Student s two-tailed t-test). (d) Frequency of CD13 + CD8 + and CD13 CD8 + TILs (n = 9-19) that express the indicated molecules. * P =.47 (paired Student s two-tailed t-test). (e) Expression (gmfi) of granzyme A or perforin in CD8 + TILs from CD13 lo tumors (n = 3-5) or CD13 hi (n = 7-8) tumors. NS, P value iignificant (Mann-Whitney test). (f) Survival of patients with lung adenocarcinoma from the TCGA dataset, classified on the basis of expression of ITGAE tracripts into CD13 hi tumors (upper 1 th percentile, n = 49) or CD13 lo tumors (lower 1 th percentile, n = 49). P =.148 (log-rank test). Each symbol represents an individual patient (a,e) or sample (c,d); small horizontal line indicates the mean (± s.e.m.). Data are from ten experiments (a) or nine to nineteen experiments (c,d) or thirteen experiments (e) or are representative of 1 experiments (b). Nature Immunology: doi:1.138/ni.3775
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doi:10.1038/nature10866 a b 1 2 3 4 5 6 7 Match No Match 1 2 3 4 5 6 7 Turcan et al. Supplementary Fig.1 Concepts mapping H3K27 targets in EF CBX8 targets in EF H3K27 targets in ES SUZ12 targets in ES
Department of Chemistry, Université de Montréal, C.P. 6128, Succursale centre-ville, Montréal, Québec, H3C 3J7, Canada.
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Supplementary. presence of the. (c) mrna expression. Error. in naive or
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