Freshly harvested intratumoral and nontumoral liver tissues, obtained by needle liver biopsy, were transferred to Hypothermosol™ FRS solution, and cells were recovered through mechanical disruption. Samples of whole fresh peripheral blood (100 µL/tube) and intrahepatic cell suspensions were divided into two tubes and immunostained with the following anti-human antibodies for surface markers: anti-CD45-APC/Cy7 (clone HI30, BioLegend), anti-CD3-PerCP-Cy5.5 (clone UCHT1, BioLegend), anti-CD56-BV605 (clone HCD56, BioLegend), anti-CD16-AF700 (clone 3G8, BioLegend), anti-CD8-PE/Cy7 (clone RPA-T8, BD Biosciences), anti-CD69-PE (clone FN50, BioLegend), anti-CTLA4-BV421 (clone BNI3, BioLegend), anti-PD-1-BV711 (clone EH12.2H7, BioLegend), anti-LAG3-PE (clone 3DS223H, eBioscience), anti-OX40-FITC (clone Ber-ACT35, BioLegend), anti-4-1BB-BV421 (clone 4B4-1, BioLegend), and anti-TIM3-BV785 (clone F39-2E2, BioLegend). FluoroFix™ Buffer (BioLegend) was used to fix the cells. The Zombie UV™ Fixable Viability kit was used to exclude dead cells, and fluorescence minus one controls were used to determine the positive immune cell population. Data were acquired with the BD-LSRII flow cytometer (BD Biosciences), collected using BD FACSDiva 6.3.1 software and analyzed using FCS Express 7 Flow software, using the previously described gating strategy,24 (link)
Figure S1
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Comprehensive Immune Profiling of Liver Tissues
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Other organizations : Université Grenoble Alpes, Centre National de la Recherche Scientifique, Inserm
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Variable analysis
independent variables
- Tissue type (intratumoral and nontumoral liver tissues)
- Expression of surface markers (CD45, CD3, CD56, CD16, CD8, CD69, CTLA4, PD-1, LAG3, OX40, 4-1BB, TIM3) on immune cells from peripheral blood and intrahepatic cell suspensions
- Peripheral blood samples
- Fluorescence minus one (FMO) controls
- Positive control: Whole fresh peripheral blood samples
- Negative control: Fluorescence minus one (FMO) controls to determine positive immune cell populations
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