This study included consecutive stage III and IV colorectal cancer treated by surgery for primary tumor or liver metastases at the Georges Francois Leclerc Center between April 2019 and May 2020. Written informed consent was obtained from all patients before enrolment. The hospital institutional review board approved the study in accordance with the principles of Good Clinical Practice, the Declaration of Helsinki, and other applicable local regulations. This study falls within the scope of the biobanking authorisation registered under the registration number AC-2014-2260.
MSI status was determined by using a panel of 6 genes (BAT26, BAT40, BAT25, NR21, NR22 and NR27) by Gold Fast PCR and fragment analysis via an ABI3130 sequencer. Tumours with two or more unstable markers were considered to have a MSI status, whereas those with no unstable markers were considered as MSS.
Fresh tumor tissues were collected on the day of surgery for each patient. Tumors were mechanically and enzymatically dissociated using a human tumor dissociation kit, according to the manufacturer's instructions (130-095-929, Miltenyi Biotec). In brief, tumors were cut into small pieces and transferred into gentleMACS C tubes containing the enzyme mix. The dissociation was performed using the gentleMACS Octo Dissociator with heaters and with the human tumor dissociation 37C_h_TDK_1 program. Samples were homogenized before being applied to a MACS SmartStrainer 70µM (130-110-916, Miltenyi Biotec) placed on a 50 mL tube. Filters were washed with 20 mL serum-free RPMI (L0500-500, Dutscher) and then centrifuged at 300g for 7 minutes. After complete aspiration of the supernatant, tumor cell suspensions were resuspended in RPMI and counted with trypan blue to remove dead cells. Then, 6.106 cells were put to one side for cytometry analyses and the rest of the cells were frozen in a solution of 50% Foetal Bovine Serum (FBS, Dutscher), 40% RPMI and 10% DMSO (P60-36720100, Dutscher) until further use.
Peripheral blood mononuclear cell isolation
Blood samples were also collected on the day of tumor resection for each patient. After recovering 1 mL of whole blood for cytometry analyses, peripheral blood mononuclear cells (PBMC) were isolated from the remaining whole blood by density gradient centrifugation (Lymphocyte Separation Medium, CMSMSL0101, Eurobio) with SepMate tubes (85460, Stemcell Technologies). Whole blood was transferred into Sepmate tubes at a rate of 17 mL of whole blood per tube and then centrifuged at 1200g for 10 min with an acceleration of 5 and the brake off. After removing as much plasma as possible, the phase containing the enriched PBMCs could be recovered. After washing with 45 mL PBS, centrifugation of 300g for 7 min was carried out and the PBMC pellet was resuspended in 5 mL PBS 1x for counting. Then, a final wash with 10 mL of PBS 1X was performed before cryopreservation, which consists in freezing at a rate of 8.106 cells per cryotube in a solution of 50% FBS, 40% RPMI and 10% DMSO until further use.
Ex vivo tumor assay restimulation
Tumor cell suspensions for each patient were thawed and diluted in RPMI1640 containing 10% FBS. After removing dead cells using the Dead Cell Removal Kit (Miltenyi Biotec), live tumor cell suspensions were labelled with 1µM of CellTrace Violet (CTV; C34571, ThermoFisher Scientific) following the manufacturer's instructions. Then, cells were resuspended in the AIM V medium (12055091, Fisher Scientific) at a rate of 5.105 cells per well in a 96-well round-bottom culture plate, and stimulated with coated anti-CD3 antibody (10 ng/mL, clone OKT3, BE0001-2, BioXcell), or 5 µg/mL of anti-CD3 and anti-CD28 (clone CD28-2; 302933, BioLegend) antibodies for the positive control, in the presence or absence of 10 µg/mL of antibodies blocking immune checkpoint receptors (anti-PDL1 (clone 6E11) and anti-TIGIT (clone 10A7) provided by Roche Institute). After 4 days of culture at 37°C with 5% CO2, 100 µL of supernatant were removed for each well and frozen at -20°C and replaced by 100µL of Brefeldin A (420601, BioLegend) 2X solution for 20 hours.
- Lymphoid and myeloid population identification
Antibodies for lymphoid cell analysis: Multicolour flow cytometry was performed using Beckman Coulter's custom design service and its dry coating technology, custom tubes containing anti-CD56-ECD (clone N901), anti-CD45-PECy5.5 (clone J.33), anti-PD-1-APC (clone PD1.3), anti-CD3-AA750 (Clone UCTH1), anti-CD45RA-PacBlue (clone 2H4LDH11LDB9), anti-CD8-KromeOrange (Clone B9.11) and a mortality marker DRAQ7 were produced. The following liquid antibodies were added to the custom tubes: anti-DNAM-1-FITC (BioLegend, clone TX25), anti-CD96-PE (BioLegend, clone NK92.39), anti-TIGIT-PECy7 (BioLegend, clone A15153G), anti-CCR7-BV605 (BioLegend, clone G043H7), anti-Tim-3-BV650 (BioLegend, clone F38-E2E2) and anti-CD4-BV785 (BioLegend, clone OKT4).
Antibodies for myeloid cell analysis: Multicolour flow cytometry was also performed using Beckman Coulter's custom design service and its dry coating technology, custom tubes containing anti-CD11b-FITC (clone Bear1), anti-HLA-DR-ECD (clone Immu-357), anti-PD-L1-APC (clone PDL1.3.1), anti-CD15-PacBlue (clone 80H5), anti-CD14-KromeOrange (clone RMO52) and a mortality marker DRAQ7 were produced. The following liquid antibodies were added to the custom tubes: anti-CD111-PE (BioLegend, clone R1.302), anti-CD155-PerCPCy5.5 (BioLegend, clone SKIL4), anti-CD112-PC7 (BioLegend, clone TX31), anti-CD163-APCCy7 (BioLegend, clone GHI/61), anti-CD3-BV605 (BioLegend, clone OKT3), anti-CD19-BV605 (BioLegend, clone HIB19), anti-CD20-BV605 (BioLegend, clone 2H7), anti-CD56-BV605 (BioLegend, clone HCD56), anti-Galectin-9-Biotin (Miltenyi Biotec, clone RG9-35.7), anti-Streptavidin-BV650 (BioLegend) and anti-CD45-BV785 (BioLegend, clone HI30).
Staining protocol: 100 μL of total heparinized blood or 1.106 cells of tumor cell suspension was added to each DURAClone tube containing liquid antibodies, vortexed immediately for 15s and incubated for 15 min at room temperature in the dark. Two millilitres of red blood lysis solution (VersaLyse solution, A09777, Beckman Coulter) containing 50 μL of the fixative agent IOTest 3 Fixative solution (A07800, Beckman Coulter) was added, inverted and incubated for 15 min in the dark. After centrifugation and washing with 3 mL of PBS 1X, cells were resuspended in 150 µL PBS 1X before acquisition on a CytoFLEX cytometer (Beckman Coulter). The gating strategies are described in Supplementary Figures 1 and 2.
- Lymphocyte function analysis
Using Beckman Coulter's custom design service and its dry coating technology, custom tubes containing anti-IFNγ-FITC (clone 45.15), anti-TNFα-PE (clone IPM2 (188)), anti-IL-4-PECy7 (clone MP4-25D2), anti-Foxp3-AF647 (Clone 259D), anti-IL-17A-AF700 (Clone BL168), anti-CD3-AA750 (clone UCHT1), anti-CD4-PacBlue (clone 13B8.2) and anti-CD8-KromeOrange (clone B9.11) were produced. Liquid antibodies were also used: anti-GranzymeB-PECy5.5 (ThermoFisher, clone GB11), anti-IL-2-BV650 (BioLegend, clone MQ1-17H12) and anti-CD45-BV785 (BioLegend, clone HI30).
Staining procedure: 1.106 cells of tumor cell suspension in 100 µL of RPMI1640 were stained with anti-CD45 mAb (clone HI30, BioLegend) in the dark for 15 minutes at RT. The cells were then washed twice and the pellet was resuspended with 50 µL of total heparinized blood. This solution was transferred into a DURactive 1 tube (C11101, Beckman Coulter) for 3 hours at 37°C in the dark. After activation, 25 μL of PerFix-NC R1 buffer (PerFix-NC kit, B31168, Beckman Coulter) was added on vortex and incubated for 15 min at room temperature. Then, 2 mL of PBS 1X was added, and after centrifugation the pellet was resuspended in 25 μL of FBS (Dutscher) and 300 μL of PerFix-NC R2 buffer was added. A 325 μL aliquot was transferred to a DURAClone tube containing the liquid antibody, vortexed immediately for 15s and incubated for 1h at room temperature in the dark. PBS 1X (3 mL) was added to the tubes, incubated for 5 min at room temperature in the dark before centrifugation for 5 min at 500g. After supernatant removal, the cells were resuspended in 3 mL of 1X PerFix-NC R3 buffer before another 5 min centrifugation at 500g. The pellet was dried and resuspended in 150 μL of 1X R3 buffer. Acquisition was done on a CytoFLEX cytometer. The gating strategy is described in Supplementary Figure 3.
- Analysis post re-stimulation with anti-PD-L1 and anti-TIGIT blocking antibodies
After 5 days of culture, tumor cell suspensions were harvested and washed with 2 mL of PBS 1X. After centrifugation, the pellets were resuspended with 50 µL of FBS and fixed with addition of 12.5 µL of PerFix-NC R1 buffer (PerFix-NC kit, B31168, Beckman Coulter) on vortex and incubated 15 min at room temperature. After incubation, 150 μL of PerFix-NC R2 buffer and the following fluorochrome-conjugated mAbs (Beckman Coulter) were added: anti-CD3 (clone UCHT1), anti-CD4 (clone 13B8.2), anti-CD8 (clone B9.11), anti-IFNγ (45.15) and anti TNFα (clone IPM2 (188)) and incubated for 1 h at room temperature in the dark. PBS 1X (3 mL) was added to the tubes, incubated for 5 min at room temperature in the dark before centrifugation for 5 min at 500g. After supernatant removal, the cells were resuspended in 3 mL of 1X PerFix-NC R3 buffer before another 5 min centrifugation at 500g. The pellet was dried and resuspended in 150 μL of 1X R3 buffer. Acquisition was done on a CytoFLEX cytometer. The gating strategy is described in Supplementary Figure 4.
CTVlo T cells were counted as proliferated cells. To further evaluate the proliferation of T cells, the mitotic index was calculated by dividing mitotic events by the absolute number of precursor cells based on the number of cells in each mitotic division. We counted the number of divided cells up to the fifth mitotic division based on the fluorescence intensity of CTV.
All cytometry analyses were done with Kaluza 1.3 software (Beckman Coulter).
Single cell RNAseq analysis was based on tumor data from 5 patients with colorectal cancer taken from Qian et al. . Processed count data were downloaded from the ArrayExpress database at EMBL-EBI (www.ebi.ac.uk/arrayexpress) under accession number E-MTAB-8107.
All variably-expressed genes were used to construct principal components (PCs) and PCs covering the highest variance in the dataset were selected. Clusters were calculated by the FindClusters function from the Seurat library  and visualised using the t-SNE dimensional reduction method. Gene characteristics of each cluster were selected using the FindMarkers function. Annotation of the resulting clusters to cell types was based on the expression of marker genes. First, only clusters expressing following genes were kept for further analysis: CD96, IFNG, TNF, PDCD1, CD274, HAVCR2, CD101, SLAMF6, TCF7. A new clustering was performed on these cell subsets, leading to 7 clusters. Among these clusters we kept 3 clusters expressing CD8 cells, and annotated among them exhausted, proliferating and activating cells (Figure 5A and Supplementary Fig. 5).
RNAseq COAD and READ TCGA datasets were downloaded using TCGA2stat R package . MSS status was obtained through the TRONCO R package . CMS classification was obtained using the CMScallerR package . All these data were available for 211 patients.
Statistical analyses were performed using Prism GraphPad software [not significant (ns), *, p < 0.05; **, p < 0.01; ***, p< 0.001; and ****, p < 0.0001]. Results are shown as the mean ± SD. Datasets were compared using an unpaired Mann–Whitney–Wilcoxon test. No statistical corrections were performed.