Immunological analyses
POLAND, APC
The peripheral blood taken from patients was collected into 4.9 ml tubes: K2-EDTA S-Monovette (Sarstedt, Germany). Next, peripheral blood mononuclear cells (PBMC) were isolated by density gradient centrifugation in Gradisol L (Aqua-Med, Poland) at 700 rcf for 20 minutes at room temperature. PBMC were counted in the Neubauer chamber. PBMC at a density of 1 × 10Next, the cells were washed twice with phosphate buffered saline (PBS) for 10 minutes, 700 rcf. Afterwards, intracellular labelling was performed using the following antibodies: anti-FoxP3 Pacific Blue (cat. no B90432, clone: 259D, Beckman Coulter), anti-IFN-gamma PE-CF594 (cat. no 562392, clone: B27, BD Biosciences), anti-IL-10 Brilliant Violet 786 (cat. no 564049, clone: JES3-9D7, BD Biosciences), anti-IL-17A APC-Cy7 (cat. no. 512320, clone: BL168, BioLegend), anti-IL-4 Brilliant Violet 605 (cat. no. 500828, clone: MP4-25D2, BioLegend), anti-E4BP4 PE (cat. no. 12-9812-42, clone: MABA223, Invitrogen), anti-T-bet APC (cat. no. 644814, clone: 4B10, BioLegend), anti-GATA-3 PerCP-eFluor 710 (cat. no. 44-9966-42, clone: TWAJ, Invitrogen) and anti-ROR gamma T Brilliant Violet 650 (cat. no. 563424, clone: RPA-T4, BD Biosciences). Incubation was performed at room temperature for 30 minutes in the darkness. After final wash with PBS, samples were analyzed on CytoFlex LX flow cytometer (Beckman Coulter, USA). Kaluza v2.1.1 (Beckman Coulter, USA) was used to analyze and graphically present the collected data (Gating strategy. Samples for cytokines and transcription factors were stained in separate tubes, nevertheless majority of gating is common for both (panels A and B), only gating of cytotoxic T cells differed. 