Phosflow

Isolated blood-derived CD8⁺ T-cells or IH-lymphocytes (0.5x10⁶/ml) were incubated with IL-7 (0.01–10 ng/ml) and/or IL-2 (100 ng/ml) and IL-15 (10 ng/ml, Sigma Aldrich, St. Louis, MO, USA) for 15 minutes at 37°C and phosphorylation of STAT5 (pSTAT5) was measured by flow cytometry, as described previously using the anti-pSTAT5 pY694 alexafluor 488 antibody (5μl/100μl cells, clone 47/STAT5(pY694), AB_399881, BD Phosflow, BD Bioscience) with fixation in 4% paraformaldehyde and permeabilization in 100% cold methanol [32 (link)] (average age of controls was 39 ± 13, HCV⁺ individuals was 43 ± 12). The expression level of pSTAT5 in CD8⁺ T-cell subsets was determined after cells were stained for phenotypic markers using CD45RA-APC and CCR7 antibodies (average age of controls was 35 ± 11, HCV⁺ individuals was 56 ± 10). To distinguish CD8⁺ T-cells from other IH-lymphocytes, 5μl CD8-PeCy5 was added with the pSTAT5 antibody. The autofluorescence of IH-lymphocytes is higher than blood-derived cells, and this was taken into account during data analysis [38 (link)].

After cell sorting by SdFFF, cell subpopulation concentrations were standardized to the same amount of cells in each condition. Anti-CD44, anti-LGR5, anti-CD133/1, and viability marker antibodies were added to the cells and incubated for 30 min at 4 °C and in the dark, antibody references are summarized in Table S1. The viability marker was used to exclude nonviable cells. Cells were then fixed in 4% paraformaldehyde (PFA, no. 10231622, Fischer Scientific) for 10 min at room temperature and permeabilized with Perm Buffer III (no. 558050, BD Phosflow ™, BD Biosciences, France) for 30 min at 4 °C. Next, antibodies recognizing the intracellular marker BMI-1 were added and incubated for 30 min at 4 °C in the dark (reference in Table S1). As reference controls, anti-IgG2bκ FITC, anti-IgG2bκ PE-Vio 770, anti-IgG1 PE-Vio 615, and mouse anti-IgG1κ PE isotype controls were used under the same conditions and concentrations to ensure specific recognition of our antibodies of interest and set gates (references in Table S1). Samples were analyzed by the CytoFlex LX and data analysis using Kaluza software v2.1 (Beckman Coulter, Indianapolis, IN, USA)

200 µL EDTA-blood from patients was incubated with recombinant interleukin 2 (IL-2) (37 °C, 15 min). After that, whole blood was lysed by incubation with lysis/fixation buffer (BD Phosflow, BD Biosciences, Heidelberg, Germany) (12 min, 37 °C). After washing (500× g, 5 min) cells were permeabilized in Perm Buffer III (BD Biosciences) for 30 min on ice. After washing (500× g, 5 min) cells were stained with anti-pSTAT5 (pY705) (intracellular), anti-CD3 peridinin-chlorophyll proteins (PerCP)-Cy5.5, anti-CD4 phycoerythrin (PE), and anti-CD8 Alexa Fluor488 antibodies. Antibodies were purchased from BD Biosciences. After 1 h (at room temperature in the dark) cells were washed and fixed with 200 µL PBS containing 1% formaldehyde. An unstimulated panel was used as negative control.

pSTAT5 was quantified in whole blood immediately after collection. After surface staining, red blood cells were lysed, and cells fixed and permeabilized using eBioscience FoxP3 protocol, followed by BD Cytofix and BD Phosflow (BD Biosciences), and 1 hour incubation at 4°C with anti-pSTAT5 monoclonal antibody and other intracellular markers (Supplementary Table 1). pSTAT5 was also quantified upon stimulation of surface stained purified CD4 T-cells, with increasing concentrations of recombinant IL-7 (0.1/1/10/50ng/ml) or IL-2 (2/20/100/200IU/ml) for 15 minutes at 37°C, as described [44 (link)].

OT-I Rag2^−/− splenocytes were co-cultured with irradiated C57BL/6 OVA_257−264 peptide-pulsed splenocytes (1:10 ratio) with 10 U/ml of rhIL-2 for the IL-4Rα assay and in the absence of rhIL-2 for the pSTAT6 assay. CD8⁺ T cells were harvested at 5, 24, and 48 h post primary and secondary stimulation. For surface analysis of IL-4Rα, cells were pelleted and stained with Zombie Aqua fixable viability kit (Biolegend) followed by AF488 anti-mouse CD8α (clone-53-6.7) (Biolegend) and PeCy7 anti-mouse IL-4Rα (clone-I015F8) (Biolegend) or PeCy7 rat IgG2b, k isotype control (clone RTK4530) (Biolegend). For analysis of phosphorylation of pSTAT6, CD8⁺ T cells were fixed with 2% paraformaldehyde (PFA) for 10 min at 37°C to halt phosphorylation events. Cells were washed and then permeabilized with chilled TruePhos perm buffer (Phospho-protein Fixation/Permeabilization Buffer Set) (Biolegend) at −20°C for 60 min. Permeabilized cells were then washed with PBS containing 1% fetal bovine serum and stained with PE rat anti-mouse CD8α (clone 53-6.7)(BD Biosciences) and Alexa Fluor 647 anti-mouse STAT6 (pY641) (BD Phosflow, BD Biosciences) or Alexa Fluor 647 mouse IgG1, k isotype control (BD Phosflow, BD Biosciences). Cells were acquired using the Fortessa X20 (BD Biosciences) and data analyzed using DIVA (BD Biosciences) and FlowJo (BD Biosciences) software.