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
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
Expression (normalized) Expression (normalized) PDCD1 expression (normalized) PD-1 a b 1.2 PBMC.3 2.4 Lung N-TIL. 7.3 NSCLC TIL 2.5 r =.8182 P =.5 4 8 Number of PD1 + cells 53.4 CD8 45.1 51.2 45.8 4.7 43.5 TIL lo TIL int TIL hi c PDCD1 4 r = -.1 5 7 8 9 Age (years) Male Female Adeno Sq I II III Tumor stage I Performance status Current Ex Never Smoking status TNFRSF9 4 r =.5 5 7 8 9 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
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
CD9 CD49a KLRG1 CD2L CCR7 Percentage -Log(P value) a CAST CX3CR1 c 1 75 5 3 9 135 18 255 77 17 23 287 24 89 3 99 199 23 37 39 4 5 3.5 2.5 1.5 TNF Reduced cell movement S1A1 25.5 TIMP1 S1PR1 Upregulated Cytokine G-protein coupled receptor Peptidase Downregulated -Log(P value) Downregulated Other b 35.2 54 31 52.5 29.8.8.8.4.7.7 1 * % of CD9 + 5 9.9 29.1.9 53.2 14.1 38.4 2.4 51. 15.2 19.7 48.2 14.1 44.2 3.4 54. 1.8 44.4 1.3 54.2 1.3 1 * % of CD49a + 5 15.4 35.9 2.3 55.8. 2.3 3.4 53 25 28.5 41.2 2.8 41.5 1.8 53.3.5 43. 4.9 53.8 1.5 1 * % of KLRG1 + 5 7.4 31.1.9 4.9 17.5 34.8 3.2 44.5 14.7 42.4 54 11.3 41. 3 5.1 1.1 38. 3.4 55 2.8 1 * % of CD2L + 5 19.4.7 2. 7 1.5 17.1 4.2.7 8.3 8.8 38 1.4 47.4.9 48.5.4 47.7.8 4.1 1.8 15 % of CCR7 + 1 5 CD8 + CD13 - TIL 17.4 CD13 2.9 13.3 2.9 21.8 8 3 8.7 3 7.4 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
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
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
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