Method Details

IHC was performed on archived FFPE tumor tissues using Ventana Benchmark and Discovery automated platforms. Briefly, after deparaffinisation and epitope retrieval in CC1 buffer (pH = 8, 36 min at 95°C), tissue sections were incubated with primary mAb for ICAM-1 (clone N1C2, GeneTex, 1:200 dilution) or E-cadherin (clone EP6, Bio SB, prediluted) for 1 h at room temperature. Amplification and detection steps used the ultraview kit with amplification, and 3,3′-diaminobenzidine was used as a chromogen. ICAM-1 staining was evaluated as H-score (0 to 300) based on the percentage of tumor cells stained at each intensity (scale from 0 to 3). Since the intensity of E-cadherin staining was homogeneous within each individual tumor sample, we scored E-cadherin staining as the prominent intensity for each tumor on a semiquantitative scale from 0 to 3.

Multiplexed fluorescent IHC for CD8 T_RM was performed by sequential staining of a single tissue section with anti-CD8 (clone SP16, Spring Bioscience, 1:200), anti-CD103 (clone EPR4166-2, Abcam, 1:200) and anti-cytokeratin (clones AE1/AE3, Agilent, 1:100). For each staining, the HRP-conjugated amplification system was associated with a tyramide-coupled fluorophore: Opal 690, Opal 250 and Opal 570, respectively. Multispectral fluorescent images were captured using the Vectra 3 microscope (PerkinElmer) and regions of interest were selected. Image analysis using InForm software (PerkinElmer) included spectral unmixing, tissue segmentation (stromal versus epithelial areas) using a trainable classifier, nuclei detection based on dapi staining and cell segmentation followed by cell phenotyping for identification of cell populations defined by the combination of individual markers. The density (number of cells per square mm) of CD8⁺, CD103⁺ CD8⁺ and CD103^-CD8⁺ cells was determined for each tumor sample in the total tumor area, as well as in the stromal and intraepithelial compartments, based on tissue segmentation. Results from image analysis were validated for all cases.

Freshly resected lung tumors and adjacent healthy lung tissue samples were immediately cut into small fragments and digested for 40 min at 37°C using the tumor dissociation kit (Miltenyi Biotech). The dissociated samples were smashed on 100 μm cell strainers, washed, and red blood cell lysis was performed. CD8 T lymphocytes were positively selected using CD8 microbeads according to the manufacturer’s instructions (Miltenyi Biotec). Recovered cells were either used for phenotypic analyses or further sorted by BD FACSAriaIII or BDFusion cell sorter (BD Biosciences) using anti-CD8-Pacific blue (RPA-T8, Biolegend), anti-CD103-FITC (Ber-ACT8, Biolegend) and anti-KLRG1-PE (clone 13F12F2, ebioscience) mAb. Dead cells were excluded using DAPI. CD103⁺CD8⁺ and KLRG1⁺CD8⁺ T cell populations were isolated and then either stored at −80°C for further DNA or RNA isolation or cultured for 2-5 days in the presence of low doses of IL-2 (20 U/ml) for functional studies. Tumor cells were recovered from the negative fraction of the CD8 T cell isolation described above, and then purified using a human tumor cell isolation kit (Miltenyi Biotec).

Total RNA was extracted from each sorted CD103⁺CD8⁺ and KLRG1⁺CD8⁺ TIL population pair and autologous healthy lung CD103⁺CD8⁺ lymphocytes when available; 150,000 lymphocytes per sample were processed using a single cell RNA purification kit (Norgen) according to the manufacturer’s instructions. RNA integrity (RNA Integrity Score ≥ 00007.0) was checked on the Agilent 2100 Bioanalyzer and quantity was determined using Qubit (Invitrogen). The SureSelect Automated Strand-Specific RNA Library Preparation Kit was used according to the manufacturer’s instructions with the Bravo Platform. Briefly, 50 ng of total RNA sample were used for poly-A mRNA selection using oligo(dT) beads and subjected to thermal mRNA fragmentation. The fragmented mRNA samples were subjected to cDNA synthesis and were further converted into double-stranded DNA using the reagents supplied in the kit, and the resulting dsDNA was used for library preparation. The final libraries were bar-coded, purified, pooled in equal concentrations and subjected to paired-end sequencing on a HiSeq-2000 sequencer (Illumina). Fast quality was assessed using FastQC (v 0.11.3) and did not require further trimming or adaptor removal. Counting of reads over the transcriptome was performed over Gencode (v 19, GRCh37.p13) with Salmon55 (v 0.8.2), using non-oriented library, 100 bootstrap and sequence bias correction. All other parameters were left to default. The pseudo-mapping rates were between 79% and 88% of overall reads. Differential analysis was performed within the R (v 3.2.3) environment with sleuth56 (v 0.28.1), wasabi (v 0.1) and in-house scripts. PCA highlights inter-individual variations that are far above any variation of interest, which was confirmed by a likelihood-test ratio and taken into account within the Wald test.

Primary functional analyses were performed using Ingenuity Pathway Analysis and GSEA was performed with clusterProfiler57 (v 3.8.0). RNaseq are available at the European Molecular Biology Laboratory European Bioinformatics Institute database (https://www.ebi.ac.uk/arrayexpress). TCR repertoire analysis was performed with a MiXCR package58 (v 2.1.10). KEGG pathway: https://www.genome.jp/kegg-bin/show_pathway?hsa04660.

For TCRseq, total DNA from CD103⁺CD8⁺ and KLRG1⁺CD8⁺ TIL subset pairs and autologous healthy lung CD103⁺CD8⁺ cells when available, sorted from nine NSCLC patient tissues, was purified using a QIAamp DNA micro kit (QIAGEN). DNA was quantified with fluorescence-based measurement Qubit (Life Technologies). TCRβ-CDR3 sequencing was performed by ImmunoSEQ, Adaptive Biotechnologies (Seattle). Raw data of TCR reads and sequences were uploaded on the ImmunoSEQ Analyzer Platform (Adaptive Biotechnologies).

For quantitative (q) RT-PCR, total RNA was extracted from sorted cell populations using the single cell RNA purification kit (Norgen Biotek). cDNA were synthesized using the Maxima first strand cDNA synthesis kit (Thermo-Fischer Scientific). qRT-PCR was performed on a step-one plus (Applied Biosystems) using Maxima SYBR Green Master Mix (ThermoFischer Scientific). Expression levels of transcripts were normalized to 18S housekeeping gene. PCR primers for human, TCF7, IKZF3, AHR, RORC, IL17 and 18S genes were designed by Sigma-Aldrich and used according to the manufacturer’s recommendations (Table S10).

Phenotypic analyses were performed by direct immunofluorescence with a panel of fluorochrome-conjugated antibodies. Anti-CD3-Alexa700 (UCHT1), anti-CD8-PacificBlue (RPA-T8), anti-CD69-APC-Cy7 (FN50), anti-granzyme-B-FITC (GB11), anti-TNFα-PE/Dazzle594 (Mab11), anti-Aiolos-Alexa488 (16D9C97), anti-Vα7.2-APC-Cy7 (3C10), anti-CD161-Alexa488 (HP-3G10), anti-TCF-1-PE (7F11A10), anti-4-1BB-PE-Dazzle594 (clone 4B4-1) and anti-TCRγδ-PerCPCy5.5 (B1) were supplied by BioLegend. Anti-CD103-BV711 (Ber-ACT8), anti-IFNγ-APC (B27) and anti-Hobit-Alexa647 (Sanquin-Hobit/1) was purchased from BD Biosciences, anti-KLRG1-PE (13F12F2), anti-CD49-PerCPefluor710 (TS2/7), anti-S1PR1-APC (SW4GYPP), anti-PD-1-PeCy7 (eBioJ105), anti-AHR-PerCPefluor710 (FF3399), anti-Ki67-PerCPefluor710 (20Raj1) were supplied by Thermo Fisher Scientific. Anti-CD45RA-APC, anti-pSTAT3pS727-APC, anti-CD39-APC, anti-IL17A-PeVio770 and anti-Tbet-APC (REA102) were purchased from Miltenyi. For intracellular expression of IFNγ, TNFα and IL-17A, cells were stimulated for 4 h with PMA (50 ng/ml) plus ionomycine (1 μg/ml) in the presence of BrefeldinA (1 μg/ml, ebioscience). Cells were fixed, permeabilized (FoxP3 buffers Kit, ebioscience) and then stained with fluorochrome-conjugated mAb. Dead cells were excluded using a LIVE/DEAD Fixable UV dead cell stain kit (Thermo Fisher Scientific). Stained cells were analyzed by flow cytometry using a BD FACS Fortessa flow cytometer (BD Biosciences). Data were processed using FlowJo V10 software (Tree Star Inc.).

For ELISA, sorted CD8⁺ T lymphocytes were stimulated overnight with PMA plus ionomycine, and then supernatants were stored until IL-17 dosage (eBiosciences).

Th17-positive control cells were generated from healthy donor’s CD4⁺ peripheral blood lymphocytes (PBL) stimulated with anti-CD3 and anti-CD28 in the presence of TGF-β and IL-6.

Formation of stable conjugates between T cells and autologous tumor cells was analyzed by confocal microscopy. Effector and target cells were co-cultured for 30 min at 1:1 E:T ratio, and then plated on poly-(L-lysine)-coated coverslips (Sigma-Aldrich, Saint-Louis, MO). Cells were then fixed, permeabilized as described16 and stained with mouse anti-phospho-tyrosine (PY20, BD Biosciences) and rabbit anti-CD8 (Thermo Fisher Scientific), followed by anti-mouse AlexaFluor-488 and anti-rabbit AlexaFluor-647 (Thermo Fisher Scientific). Coverslips were mounted and analyzed using a fluorescence microscope (Leica, HR Sp8) with x63 lenses, and polarization of phospho-Tyr to the immune synapse between T cells and target cells was calculated. Stable conjugates were defined by polarization of p-Tyr at the contact zone between effector cells and tumor cells. Cytotoxic activity was evaluated using the conventional 4 h ⁵¹Cr-release assay as described.16