Facsfortessa 2

PBMCs were stimulated with positive control (S. aureus peptidoglycan) or mock control or with the different gp120 constructs. Cells were washed in PBS containing 2% FBS and 2 mM EDTA, blocked with 10% normal human serum for 15 min at 4°C, and then stained for CD3, CD14, and CD19 (1 μL of each antibody in 100 μL of PBS containing 2% BSA and 2 mM EDTA) for 30 min at 4°C in the dark. Dead cells were excluded using Zombie Aqua Dye (BioLegend). For intracellular staining of IL-10 and TNF- α, Brefeldin A was added 6 hours post-stimulation and cells were cultured overnight. Then, cells were stained for surface markers before being fixed, permeabilized and stained for IL-10 and TNF-α. For intracellular cytokine staining of IL-1, a mix of Brefeldin A, PMA and ionomycin was added 12 hours post-stimulation with gp120 during 4 hours and cells were then fixed and permeabilized for staining. Events were collected on a FACSFortessa II (BD) and analyzed using FlowJo software (Tree Star Inc.).

For intracellular staining, isolated gut and skin lymphocytes were counted for viable cells (1 × 10⁶) and stimulated with PMA (50 ng/mL) and ionomycin (1 μg/mL) for 3 h at 37 °C. Cells were further incubated for 2 h with Golgi stop solution (containing Monesin, final concentration 1 µM), washed with PBS, before surface antigens staining with Thy1.2 (BD, Pharmingen, Clone: 30-H12) and streptavidin conjugated linear markers; NK1.1 (Biolegend, Clone PK136), TCRγδ (BD pharmingen, Clone: GL3), CD11b (BD Pharmingen, Clone: M1/70), CD8α (BD Pharmingen, Clone: 53–6.7), CD19 (ebioscience, Clone: eBio1D3), GR-1 (BD, Clone: RB6-8C5), and Ter119 (BD Pharmingen, Clone: Ter-119) at 4 °C for 15 min. Cells were then incubated with fixation solution (Cytofix, BD) at 4 °C for 50 min, washed twice and stained with intracellular antibodies; RORγt (BD Horizon, Clone: Q31-378), Gata3 (BD Pharmingen, Clone: L50-823), IL-5 (Biolegend, Clone: TRFK5), IL-13 (ebioscience, Clone: eBio13A), IL-17A (Biolegend, Clone: TC11-18H10.1), and IL-22 (Biolegend, Clone: Poly5164) antibodies at 4 °C in the dark for 30 min. Cells were analysed using FACS Canto II or FACS Fortessa II (BD, Biosciences). Cells sorting was performed using FACS Aria III cell sorter (BD, Biosciences). Flow cytometry data were analysed by FlowJo software (version 10.7.1; https://www.flowjo.com/solutions/flowjo).