Ebioscience fixation permeabilization concentrate and permeabilization buffer

For detailed analysis of the adoptively transferred CD4⁺ T cells, red blood cells from TC were eliminated by using ACT buffer for 6 minutes at RT with constant shaking. Upon washing in PBS (Thermo Fisher Scientific), remaining cells were fixed and permeabilized using eBioscience™ fixation/permeabilization concentrate and permeabilization buffer (Thermo Fisher Scientific) according to the manufacture’s description. After block of the Fc receptors, TC cells were stained with combinations of fluorophore (APC, FITC, PE, PE-Cy7, PerCp.Cy5.5)-conjugated anti-mouse CD4 (clone RM4-5), IFN-γ (clone XMG1.2), IL-4 (clone 11B11), IL-10 (clone Jes5-16E3) and IL-22 (clone Poly5164) monoclonal antibodies from Biolegend and IL-5 (clone TRFK5) and IL-17A (clone eBio17B7) monoclonal antibodies from Thermo Fisher Scientific. Samples were acquired using the FACS Canto I (BD Bioscience) or the CytoFlex S cytometer (Beckman Coulter, Krefeld, Germany) and compensation was performed using BD™ CompBead particles (BD Biosciences) or the VersaComp antibody capture kit (Beckman Coulter). In addition, fluorescence minus one (FMO) controls were acquired to discriminate populations. Finally, antibody expression levels were analysed using the FlowJo v10 software (FlowJo, Portland, USA). Supplementary Figure 3 shows the applied gating strategy.

To obtain whole blood cells from the in vitro cultures, plates were centrifuged and supernatants removed. Red blood cells were then eliminated from the cultures using a red blood cell lysis buffer (Biolegend, San Diego, USA) and remaining cells were fixed and permeabilized using eBioscience^™ fixation/permeabilization concentrate and permeabilization buffer (Thermo Fisher Scientific) according to the manufacture`s description. Thereafter, cells were stained with combinations of fluorophore (FITC, PE, PE-Cy7, APC)-conjugated anti-human CD1d (clone 51.1), CD4 (clone RPA-T4), CD5 (clone UCHT2), CD19 (clone HIB19), CD24 (clone eBioSN3 (SN3 A5-2H10)), CD38 (clone HIT2), CD127 (clone eBioRDR5), FOXP3 (clone 236A/E7), HELIOS (clone 22F6), IL-10 (clone JES3-9D7), eBioscience^™ monoclonal antibodies from Thermo Fisher Scientific and CD304 (Neuropilin-1, clone 12C2) monoclonal antibody from Biolegend. Stained samples were stored at 4°C and kept in the dark. Within 7 days, the samples were transported to the Kumasi Centre for Collaborative Research in Tropical Medicine (KCCR) in Kumasi, Ghana and acquired using the BD Accuri^™ Flow cytometer (BD Bioscience). Afterwards antibody expression levels were analysed using the FlowJo v10 software (FlowJo, LLC, USA). An overview of the analysed regulatory immune cell subsets with their corresponding flow cytometry markers is shown in Table 1.